EA003256B1 - METHOD FOR TREATING MALIGNANT TUMOR BY IN VIVO GENE THERAPY WITH USE OF GENES ENCODING INTERFERON-beta - Google Patents

Abstract

1. A method for treating cancer by in vivo gene therapy comprising the step of administering to a subject a therapeutically effective amount of a viral vector, characterized in that said vector comprises a gene that encodes interferon-beta, and wherein said vector is selected from the group consisting of an adenoviral vector, a lentiviral vector, an Epstein Barr viral vector, a baculoviral vector and a herpes simplex viral vector. 2. The method according to claim 1, characterized in that the vector is selected from the group consisting of an adenoviral vector, a baculoviral vector and a lentiviral vector. 3. The method according to claim 1, characterized in that the vector is an adenoviral vector. 4. The method according to claim 3, characterized in that the adenoviral vector has a deletion in its E3 gene. 5. The method according to claim 3 or 4, characterized in that the adenoviral vector has a deletion in its E1 gene. 6. The method according to claim 1, characterized in that the vector is administered by a route selected from the group consisting of direct injection into a tumor, topical administration, intraocular administration, parenteral administration, intranasal administration, intratracheal administration, intrabronchial administration and subcutaneous administration. 7. The method according to claim 6, characterized in that said route is a parenteral administration route selected from the group consisting of intravenous administration, intramuscular administration and intraperitoneal administration. 8. The method according to claim 6, characterized in that the vector is administered by direct injection into a tumor. 9. The method according to claim 1, characterized in that the tumor is selected from a group consisting of malignant glioma, melanoma, hemangioma, leukemia, lymphoma, myeloma, colorectal cancer, non-small cell carcinoma, breast cancer and ovarian cancer. 10. The method according to claim 9, characterized in that the tumor is malignant glioma. 11. A method for treating cancer by in vivo gene therapy comprising the step of administering to a subject a therapeutically effective amount of a replication-defective vector, characterized in that said vector comprises a gene that encodes interferon-beta, and wherein said viral vector is selected from the group consisting of a adenoviral vector, a lentiviral vector, a baculoviral vector, an Epstein Barr viral vector and a herpes simplex viral vector. 12. The method according to claim 11, characterized in that the vector is selected from the group consisting of an adenoviral vector, a baculoviral vector and a lentiviral vector. 13. The method according to claim 12, characterized in that the vector is an adenoviral vector. 14. The method according to claim 13, characterized in that the vector is deleted in one or more genome regions selected from the group consisting of the E1, E2A and E4 regions of the adenoviral genome. 15. The method according to claim 13 or 14, characterized in that the vector has a deletion in its E3 gene. 16. The method according to claim 14, characterized in that the vector is deficient in its E1 gene. 17. The method according to claim 16, characterized in that the vector additionally has a deletion in its E3 gene. 18. The method according to claim 11, characterized in that the vector is administered by a route selected from the group consisting of direct injection into a tumor, topical administration, intraocular administration, parental administration, intranasal administration, intratracheal administration, intrabronchial administration and subcutaneous administration. 19. The method according to claim 18, characterized in that said route is a parenteral administration route selected from the group consisting of intravenous administration, intramuscular administration and intraperitoneal administration. 20. The method according to claim 18, characterized in that the vector is administered by direct injection into a tumor. 21. The method according to claim 11, characterized in that the tumor is selected from a group consisting of malignant glioma, melanoma, hemangioma, leukemia, lymphoma, myeloma, colorectal cancer, non-small cell carcinoma, breast cancer and ovarian cancer. 22. The method according to claim 21, characterized in that the tumor is malignant glioma.

Description

This invention relates to gene therapy. In particular, the present invention relates to the introduction of DNA encoding secreted proteins, such as interferons, to humans and animals.

BACKGROUND OF THE INVENTION

Interferons (also called ’’) are proteins with different biological activities, in particular antiviral, immunomodulatory and antiproliferative. Interferons are relatively small, species-specific, single-chain polypeptides produced by mammalian cells in response to various inducers, such as viruses, polypeptides, mitogens, and the like. Interferons protect animal tissues and cells from viruses and constitute an important host defense mechanism. In most cases, interferons provide better protection for tissues and cells of the type in which they were produced, compared with other types of tissues and cells, which implies that human interferon can treat human diseases much more effectively than interferons produced by animals of other species.

There are several different types of human interferons that can be classified as leukocyte ([α] alphainterferon), fibroblast ([β] betainterferon), immune interferon ([γ] gammainterferon), and a large number of their variants. A general description of interferons can be found in various scientific articles and monographs, including: Tke 1n1sgGsgop Buk1st (^ .E. 81c \\ 'ag1. II, Brtshdeg-Ueg1ad, Ν.Υ. 1979); and 1n1sgGsgop Tkegaru (\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\: Stock Nume: 1 Veroise Bepez, 676, \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\RE RENODES OF OF THE UNIVERSAL), incorporated herein by reference.

Interferons are very promising for the treatment of a number of human malignant tumors, since these molecules exhibit anticancer activity at many levels. First, interferons can directly inhibit the proliferation of human tumor cells. Moreover, their antiproliferative activity is synergistic with the action of various proven chemotherapeutic agents, such as cis-platinum, 5EI and taxol. Secondly, the immunomodulatory effect of interferons can induce an antitumor immune response. This reaction activates ΝΚ cells, stimulates the action of macrophages and induces surface expression of MHC class I, which induces the activity of antitumor cytotoxic lymphocytes. In addition, some studies have found that β-белок protein has antiangiogenic activity. Angiogenesis, that is, the development of new blood vessels, is important for the growth of solid tumors. Available evidence suggests that β-ΙΕΝ can inhibit angiogenesis by inhibiting the expression of pro-angiogenic factors, such as LEOE and UEOE. Finally, interferons can inhibit tumor invasiveness by affecting the expression of enzymes such as collagenase and elastase, which are important for tissue remodeling.

Interferons also have an antiviral effect, which is based on two different mechanisms. For example, type I interferons (α and β) can inhibit the replication of human hepatitis B virus (HLV) and hepatitis C virus (HLV), while they can stimulate the immune response against cells infected with these viruses.

Despite the promising therapeutic value, interferons are of very limited use in clinical practice for the treatment of viral hepatitis and solid tumors. Protein αΠΕΝ is certified for use in the treatment of HBI and NSA; however, the recovery rate in both cases is approximately 20%. Although interferons are also certified for the treatment of certain types of malignant tumors, such as lymphoma, leukemia, melanoma and a malignant tumor of the kidney, in most clinical trials in which interferons were used alone or in combination with conventional chemotherapeutic agents for the treatment of solid tumors, no positive results were obtained .

The route of administration of interferon is an important factor in the clinical use of this important drug. The most common is the systemic administration of interferon in the form of intravenous, intramuscular or subcutaneous injections, while achieving some success in the treatment of diseases such as malignant reticuloendotheliosis, acquired human immunodeficiency syndrome (AIDS) and Kaposi's sarcoma. However, it is known that proteins in purified form are particularly susceptible to degradation. In particular, the primary mechanism for the destruction of betainterferon in solution is aggregation and deamidation. To date, the use of interferons has been very limited due to poor stability in solutions and other products. In addition, in the case of parenteral administration of interferon (intramuscularly, subcutaneously or intravenously), the interferon protein is very rapidly excreted from the body. Therefore, parenteral administration of this protein does not ensure the delivery of a sufficient amount of interferon to the focus of the disease (solid tumor or liver in case of hepatitis). The amount of interferon that can be administered to patients parenterally is limited due to side effects that occur when large doses of interferon are administered. From the above it follows that there is a need for more effective therapy.

SUMMARY OF THE INVENTION

This patent application aims to eliminate the problems associated with the delivery of a secreted protein, such as interferon, used as a medicine to the disease site. The present invention relates to a method for treating interferon, in accordance with which the delivery of the gene encoding the secreted protein, and not the protein itself, is performed.

Thus, an object of the present invention is a gene therapy method based on the use of genetically engineered cells and its use for delivering a secreted protein, such as interferon, to a focal point in a recipient mammal. The present invention provides the achievement of these and other goals by developing methods of forming a cellular expression system and using the resulting expression system and pharmaceutical compositions containing the specified system. The cell expression system expresses a gene encoding one or more secreted proteins and is a useful carrier for delivering the gene product to the active site of the mammalian recipient. In accordance with a preferred embodiment of the invention, the recipient mammal is a human.

One embodiment of the invention is a cell expression system for expressing ίη νίνο in an interferon recipient mammalian cell for treating a particular disease. The expression system includes a cell of the same species as the recipient mammal and an expression vector introduced therein to express interferon. The recipient mammal is preferably a human, and the expression vector is a viral vector.

In accordance with yet another embodiment of the invention, the expression system comprises a plurality of cells of the same species as the mammalian recipient and an expression vector inserted therein for expressing a secreted protein. The expression vector is located only in part of the cells. It preferably contains at least 0.3% of all cells. The preferred secreted protein is interferon and the most preferred interferons are alpha, beta, gamma and consensus interferons, with betainterferon being the most preferred.

In accordance with other variants of the invention, the cell expression system includes many malignant cells, and at least part of the malignant cells contains an adenoviral vector with an isolated polynucleotide encoding interferon during expression. The adenoviral vectors used in this cellular expression system are selected from the group consisting of: (a) an adenoviral vector with a deletion and / or mutation in the E1 gene; (B) an adenoviral vector with a deletion and / or mutation in the E2a gene, wherein said vector expresses human beta-interferon; (c) an adenoviral vector with a deletion and / or mutation in the E1 and E4 genes; and (b) an adenoviral vector with a deletion of all genes, said vector expressing human beta-interferon.

A method for gene therapy of ίη νίνο includes the step of introducing an adenoviral vector containing an isolated polynucleotide encoding a human (β) beta-interferon directly into a cell of a subject without first removing said cell. In accordance with the methods of ίη νίνο and ex νίνο, the drug can be used for local, intraocular, parenteral, nasal, intratracheal, intrabronchial, intramuscular, subcutaneous, intravenous and intraperitoneal administration.

The present invention has several advantages. Gene therapy with interferon allows you to create very high local concentrations of interferon at low system levels. This provides higher efficacy with less pronounced side effects. In addition, interferons delivered to the disease focus by gene therapy are kept at a fairly constant level unlike other interferon treatments, in which the very high bursts or peaks in the concentration of interferon (which can cause toxicity) that occur after protein injection are replaced by very low levels when the concentration of interferon is apparently too low to have an effective effect.

The convenience of the subject is also important. This method allows you to replace frequent injections of interferon with just one or more rare injections of a vector expressing the interferon gene, which provides long-term sustainable protein production. Gene therapy allows you to control the delivery of interferons to a specific target organ or tumor. An autocrine system can be created in which the same cells express, secrete and absorb interferon. So very high local doses can be achieved. Such high doses cannot be obtained by parenteral administration of the protein due to toxicity problems.

Since cells secrete interferon proteins, it is not necessary to transduce the interferon genome of all tumor cells or hepatitis-affected liver cells. Cells that do not absorb the indicated gene will be under the influence of neighboring cells (the so-called imitation effect), which have this gene and secrete interferon. This important discovery, apparently, should have a strong influence on treatment regimens, since now it is not necessary that all cells in the tumor formation or in the affected organ contain an expression vector.

And finally, it was found that parenteral administration of interferon causes the formation of antibodies against interferon. It is possible that this humoral immune response, potentially neutralizing the effect of the drug, can be reduced by introducing the interferon gene into a specific local area. In addition to local expression, expressed interferon can be produced by endogenous human cells, therefore it will be more natural in structure and glycosylation and possibly less immunogenic than interferon produced by bacteria, yeast or Chinese hamster ovary cells, and then purified and introduced as parenteral injections.

These and other objectives of the present invention, as well as its advantages and methods of application, are apparent from the detailed description of the preferred embodiments and the accompanying drawings.

Brief Description of the Drawings

FIG. 1. (A) Uninfected ΜΌΑ-ΜΒ-468 (-O-) cells or these cells infected with Η5.110ΗΙΕΝβ cells in an amount of 0.01% (-Δ-), 0.03% (- □ -), 0, 1% (-V-) or 0.3% (- © -), were injected subcutaneously into the side of the mice, and after implantation of the tumor cells, a graph of the change in the average tumor size versus time was plotted; (B) Kaplan-Meir graph showing the percentage survival of mice over the entire observation period of 109 days. Uninfected cells (-O-); cells infected with H5.1101ιΙΡΝβ in an amount of 0.01% (-Δ-), 0.03% (- □ -), 0.1% (-V-) or 0.3% (- © -).

FIG. 2. (A) Gene therapy with beta-interferon ex νίνο using (1) KM12B4A cells; (2) Niy7 cells; and (3) ME180 cells. A graph of the average tumor size versus time is plotted after the implantation of the tumor cells. Uninfected cells (-) or cells infected with Η5.110ΜΕΝβ in the amount of 1% (- • -) or 10% (- ▲ -) were implanted into mice. In those groups where some mice were euthanized, the size of the tumor is presented as an average value, with the last value obtained for euthanized animals being indicated first. Interruption of schedules corresponds to the death or mortification of all animals in the group. (B) Percentage of survival of mice over the entire observation period of 70 days. Blocks 1, 2, and 3 show data obtained for mice that were respectively implanted with KM12B4A cells, Niy7 cells, and ME180 cells. The following symbols indicate mice implanted with uninfected cells (-); cells infected with H5.1101ιΙΡΝβ in an amount of 1% (- • -) or 10% (- ▲ -).

FIG. 3. Direct treatment of ίη νίνο tumors MEA-MV-468. H5.1101ιΙΕΝβ was injected into the tumor in an amount of, respectively, ZxU ⁹ PFU (plaque forming units) (- © -), 1x10 ⁹ PFU (-BE-), 3x10 ⁸ PFU (-O-), 1x10 ⁸ PFU (Δ-) and 3x10 ⁷ PFU (-V-); saline with phosphate buffer (PB8) (-B-) or

Η5.1101αοΖ in the amount of, respectively, 3x10 ⁹ PFU (-◊-), 1x10 ⁹ PFU (- ▲ -), 3x10 ⁸ PFU (-X-) and 1x10 ⁸ PFU (-> -). Tumor sizes were measured within 14 days after injection.

Description of preferred embodiments of the invention

The present invention is based in part on the development of an ex νίνο gene therapy method, the implementation of which uses a cell that (1) can be easily removed from the patient's body; (2) can be modified ίη νίΐτο by introducing genetic material; (3) can be implanted into the recipient; (4) is not thrombogenic and (5) can be implanted into the recipient in large quantities. In the генη νίνο gene therapy method under consideration, a cell is used that is (1) located in the recipient’s body and (2) can be modified with ίη Shea to express isolated genetic material. A genetically modified cell includes regulatory elements that allow you to control the amount of expressed genetic material.

Genetically modified cells must remain viable and continue to produce the expressed material ίη δίΐπ for the period of time necessary for the expressed material to have a beneficial (i.e. therapeutic) effect, without interfering with the normal functioning of the tissue in which these cells are located.

The expressed material is preferably a secreted protein (as defined below). The secreted protein is most preferably interferon. Although interferons are the most preferred therapeutic agent, a general principle has been identified that is applicable to gene therapy with any secreted protein. We have found that since secreted proteins, such as interferons, are isolated from the cells in which they are expressed, it is not necessary to transduce the secreted protein into all cells of the tumor or, for example, a hepatitis-affected liver. Cells that do not absorb this gene will be influenced by neighboring cells that have such a gene and secrete a protein (i.e. the so-called imitation effect). Although these gene therapy methods are described for isolated polynucleotides that encode interferons during expression, any secreted protein (defined below) can be used in the methods and compositions of the present invention.

All references cited in this application are hereby incorporated by reference.

I. Definitions.

Gene therapy means a technique that allows you to adjust the phenotype of the disease by introducing genetic information into the affected body.

Gene therapy ex νινο means a technique according to which cells are removed from a subject and their cultivation is ίη νίίτο. A polynucleotide, such as a functional gene, is introduced into these ίη νίίτο cells, after which the modified cells are propagated in culture and implanted again to this subject.

Gene therapy ίη νινο means a technique according to which target cells are not removed from a subject. Instead, the isolated polynucleotide (for example, a gene encoding interferon during expression) is introduced into the cells of the recipient ίη δίίιι. that is, into cells located in the recipient's body. Ίη νινο gene therapy has been studied in several animal models, and recent publications report the possibility of direct transfer of the ίη δίίιι gene to organs and tissues.

Genetically treatable conditions mean conditions such as genetic diseases (i.e., a disease caused by defects in one or more genes), acquired pathologies (i.e., pathologies that are not caused by a birth defect), malignant diseases, and preventative procedures (i.e., preventing a disease or unwanted medical condition).

Acquired pathology means a disease or syndrome manifesting itself in an inappropriate physiological, biochemical, cellular, structural or molecular biological state.

Polynucleotide means a polymer consisting of monomeric units of a nucleic acid, said monomeric units being ribonucleic acids (RNA), deoxyribonucleic acids (DNA), or combinations thereof. The four bases of DNA are adenine (A), guanine (C), cytosine (C) and thymine (T). The four bases of RNA are A, C, C and uracil (I).

Isolated with respect to polynucleotide sequences of genes encoding interferon, means an RNA or DNA polynucleotide, part of a genomic polynucleotide, cDNA or synthetic polynucleotide, which due to its origin or manipulation: (ί) is not associated with all the polynucleotide with which it is associated in nature (for example, present in the host cell as part of an expression vector); (ίί) linked to a nucleic acid or other chemical moiety that differs from that moiety in nature; or (w) not found in nature. The term isolated hereinafter means a polynucleotide sequence that: (ί) is amplified by ίη νίίτο, for example, by polymerase chain synthesis reaction (CSC); (ίί) synthesized by a chemical method; (w) produced by recombinant method by cloning; or (ίν) is purified by digestion and gel separation.

Thus, an isolated interferon polynucleotide includes, for example, a non-natural nucleic acid that can be transcribed into antisense RNA, as well as a gene encoding interferon that is not expressed or expressed at biologically insignificant levels in a natural cell. To illustrate the foregoing, it can be noted that a synthetic or natural gene encoding human betainterferon 1a should be considered isolated in relation to human brain cells, since these cells do not express interferon 1a in vivo. Another example of an isolated polynucleotide is the introduction of only part of the interferon gene to create a recombinant gene, in particular combining an inducible promoter with a sequence encoding endogenous interferon by homologous recombination.

A gene means a DNA sequence (i.e. a linear matrix of nucleotides connected to each other by 3'-5'-terminal pentose phosphodiester bonds), which encodes the amino acid sequence of a specific protein using mRNA.

Transcription means the process of synthesizing mRNA from a gene.

Translation means the process of synthesizing protein from mRNA.

Expression means a process undergoing a DNA or gene sequence for protein synthesis, combining transcription and translation.

Inhibition of growth, as used herein, this term refers to both inhibition of growth of a target cell (i.e., a tumor) and inhibition of a transformed phenotype (as measured, for example, by changes in morphology).

Amino acid means a monomeric unit of a peptide, polypeptide or protein. There are twenty b-isomers of amino acids. This term also includes amino acid analogues and protein amino acid isomers and their analogues.

Protein means any polymer consisting of virtually any 20 amino acids of a protein, regardless of size. Although the term polypeptide is often used to define relatively large polypeptides and the term peptide is often used to define small polypeptides, these terms are used interchangeably. The term protein, as used herein, refers to peptides, proteins, and polypeptides, unless otherwise indicated.

Secreted protein means a protein transported from a cell to the outside; among secreted proteins there are a large number of growth factors and immunomodulating proteins, in particular various interferons (α, β, γ), interleukins, such as 1B-1, -2, -4, -8 and -12, and growth factors, such like SM-S8R, SS8R.

Genetic fusion means the colinear, covalent binding of two or more proteins through their peptide backbones by genetic expression of a polynucleotide molecule encoding these proteins.

Mutant means any qualitative or structural change in the genetic material of an organism, in particular any change (i.e., deletion, substitution, addition or modification) in the wild-type interferon gene or any change in the wild-type interferon protein.

Wild type means the natural polynucleotide or amino acid sequence of an interferon gene or protein as it exists ίη νίνο.

Standard hybridization conditions mean salt production conditions and temperature conditions, which are usually set for hybridization and washing from 0.5 X 88C to about 5x88C and 65 ° C. The term standard hybridization conditions, as used herein, is a working definition and covers the entire range of hybridization conditions. More stringent conditions may, for example, include hybridization using a plaque screening buffer (0.2% polyvinylpyrrolidone, 0.2% 400, 0.2% bovine serum albumin, 50 mM Tris-HCl (pH 7.5), 1M NaCl, 0.1% sodium pyrophosphate, 1% 8-8); 10% dextran sulfate and 100 μg / ml denatured, sonicated salmon sperm DNA at a temperature of 65 ° C for 12-20 hours and washing with 75 mM NaCl / 7.5 mM sodium citrate (0.5x88C) / 1% 8 %8 at temperature 65 ° С. Less stringent conditions may, for example, include hybridization using plaque screening buffer, 10% dextran sulfate and 110 μg / ml denatured, sonicated salmon sperm DNA at 55 ° C for 12-20 hours and washing with 300 mM BaCl / 30 mM sodium citrate (2.0x88C) / 1% 8Ό8 at a temperature of 55 ° C.

An expression control sequence means a nucleotide sequence that controls and regulates gene expression upon operative binding to these genes.

Operatively linked means a polynucleotide sequence (DNA, RNA) that is operatively linked to an expression control sequence when said sequence controls and regulates the transcription and translation of a given polynucleotide sequence. The term operatively linked includes the presence of an appropriate initiating signal (e.g., ATS) in front of the expressed polynucleotide sequence and maintaining the correct reading frame for the expression of the polynucleotide sequence under control of the expression control sequence and producing the desired interferon encoded by the selected polynucleotide sequence.

An expression vector means a polynucleotide, typically a DNA plasmid (which also includes a virus), which allows expression of at least one gene when an expression vector is introduced into a host cell. This vector may or may not be able to replicate in the cell.

Tumor means any unwanted cell proliferation. Such cell growth includes malignant and benign, solid or liquid tumors, carcinomas, myelomas, sarcomas, leukemia, lymphomas, and other malignant, tumor, or oncogenic diseases.

A genetically modified cell (also called a cell expression system) includes a cell and an expression vector for expressing interferon. If ex νίνο is used, genetically modified cells must be suitable for administration to a recipient mammal, where they replace or coexist with endogenous recipient cells. When using ίη νίνο, such cells are produced in the recipient's body.

The present invention also relates to various methods for the preparation and use of the above genetically modified cells. In particular, this invention relates to a method for the genetic modification of the cell (s) of a recipient mammal ex νίνο and the introduction of genetically modified cells to a recipient mammal. According to a preferred embodiment of the invention for ex νίνο gene therapy, said cells are autogenous cells, that is, cells removed from a recipient mammal. As used herein, the term “deleted” means a cell or a plurality of cells that have been removed from their natural location ίη νίνο. Methods of removing cells from a patient and methods of maintaining isolated cells in culture are well known to those skilled in the art (δίνΐίαηοιι. E., e1 a1., Kk1pe \ Ιηί1. 37: 1563-1570 (1992); H) e11e, 1.N., e1 a1., reps; ·]! Όί; · ι (ν5ί5 Ιηίί. 9: 341-347 (1989); Ne1Bsh, R., Vyusesh.D.283: 165-170 (1992); Όί Ραο1ο, N .. e1 a1., Ιηΐ. 1. Ay. Sep ., 12: 485-501 (1989); Όί Ραο1ο, Ν., E1 a1., Syssa1 No. Ryugo., 34: 179-1848 (1990); Όί Ραο1ο, Ν., E1 a1., No.ρΗτοη., 57 : 323-331 (1991)). All patents, patent applications, and publications cited above and below in the Detailed Description of the Invention are incorporated herein by reference.

ΙΙ. Isolated polynucleotides of interferon.

Interferon (also referred to as ΙΕΝ) is a relatively small, species-specific, single-chain polypeptide produced by mammalian cells in response to various inducers, such as viruses, polypeptides, mitogens, and the like. The most preferred interferons are in recombinant form, and methods for producing proteins based on recombinant DNA, including various interferons, are well known and do not limit the scope of this invention. See, for example, US Patent Nos. 4,399,216, 5,149,636, 5,179,017 (Ax1 e1 a1.) And 4,470,461 (CaiGshan). Recombinant forms of alpha, beta, gamma and consensus interferon were obtained. Different forms of interferon can be expressed in cells containing polynucleotide sequences encoding variants such as mutants with eliminated cysteine (for example, for beta interferon) or mutants with eliminated methionine. Other modifications can be obtained using post-translational processing systems of the host cell. Therefore, the exact chemical structure of a particular interferon will depend on several factors and does not limit the scope of this invention. All interferons that are part of the compositions described herein retain their biological activity under acceptable environmental conditions.

Preferred polynucleotides that can be used in the methods of the present invention are isolated from wild-type interferon gene sequences from different vertebrates, preferably mammals, and obtained by methods well known to those skilled in the art. See, for example, US Patent No. 5,641,656 (issued June 24, 1997; DNA encoding avian interferon type типа protein and mature avian interferon type Ι), US Patent No. 5,605,688 (February 25, 1997; recombinant dog and horse type фер interferons ); US patent No. 5554513 (September 10, 1996; DNA sequence encoding human beta-interferon 2A); US patent No. 5541312 (July 30, 1996; DNA encoding the human fibroblast beta-2 interferon polypeptide); US patent No. 5231176 (July 27, 1993; a DNA molecule encoding human leukocyte interferon); US patent No. 5071761 (December 10, 1991; a DNA sequence encoding a subsequence of human lymphoblastoid interferons L2Nl-2 and LlNl-3 human); US patent No. 4970161 (November 13, 1990; DNA sequence encoding human gamma interferon); US patent No. 4738931 (April 19, 1988; DNA containing the human beta-interferon gene); US patent No. 4695543 (September 22, 1987; Cx-1 gene of human alpha interferon) and US patent No. 4456748 (June 26, 1984; DNA encoding the subsequences of different natural leukocyte interferons).

In accordance with the present invention, mutants of interferon family genes can be used. Mutations of the polynucleotide sequence of wild-type interferon are obtained using conventional directed mutagenesis methods that are well known to those skilled in the art. The term mutant also means genetic fusion, therefore, all of the following interferon sequences, incorporated by reference into this specification, should be considered mutant sequences:

US patent No. 5273889 (December 28, 1993; DNA construct containing the gamma interferon gene associated with the sequence encoding the immunogenic leukotoxin); US patent No. 4959314 (September 25, 1990, a gene with a DNA sequence encoding a synthetic mutein of a biologically active native protein); US patent No. 4929554 (May 29, 1990; DNA encoding the recombinant immune interferon Be5-SU8TUK.-CT8 person); US patent No. 4914033 (April 3, 1990, a DNA molecule encoding a modified beta interferon comprising beta interferon) and US patent No. 4569908 (February 11, 1986; DNA having a nucleotide sequence that encodes a multifunctional hybrid interferon polypeptide )

In addition, the isolated polynucleotides described in these patents can be rearranged to obtain functionally equivalent polynucleotides. A polynucleotide is functionally equivalent compared to polynucleotides of the above sequences if it satisfies at least one of the following conditions:

(a) the functional equivalent is a polynucleotide that hybridizes to any of the above sequences under standard hybridization conditions, and / or is degenerate with respect to any of the above sequences. Most preferably, it encodes a mutant interferon having the therapeutic effect of wild-type interferon;

(b) the functional equivalent is a polynucleotide that encodes in the process of expression the amino acid sequence encoded by any polynucleotides of the above interferon sequences.

In short, the term interferon includes, but is not limited to, all of the above substances, as well as their functional equivalents. As used herein, the term functional equivalent means an interferon protein or polynucleotide encoding an interferon protein that has the same or better effect on a recipient mammal as interferon, the functional equivalent of which it is. As is well known to any person skilled in the art, functionally equivalent protein can be obtained by recombinant methods, for example, by expression of functionally equivalent DNA. Therefore, the scope of the present invention includes interferons encoded by natural DNA, as well as non-natural DNA, which encode the same protein as encoded natural DNA. Due to the degeneracy of the nucleotide encoding the sequence, other polynucleotides can be used to encode interferons. These include all or parts of the above sequences, which are changed by replacing different codons encoding the same amino acid residue in the sequence, resulting in a silent mutation. For example, Pye (P) is encoded with two codons, TTC or TTT, Tug (Υ) is encoded TAC or TAT, and Ηίδ (Η) is encoded CAC or CAT. On the other hand, Tgr (ED) is encoded by one codon, TCC. Therefore, it is obvious that for a given DNA sequence encoding a specific interferon, there are many degenerate DNA sequences capable of encoding this interferon. All of these degenerate DNA sequences are included in the scope of the present invention.

Consensus interferon is also included in this definition. As used herein, the term “consensus interferon” means a non-natural polypeptide that mainly includes those amino acid residues that are common to sequences of all subtypes of natural human interferon and contain, in one or more positions where there is no amino acid common to all subtypes, an amino acid, which occurs mainly in this position, and in no case contains any amino acid residue that does not occur in this position in at least one th natural subtype. Consensus interferon sequences include the consensus sequences of any of the above interferons, provided that they have subtype sequences. Typical consensus interferons are described in US Pat. Nos. 4,695,623 and 4,897,471 (ATDEP). The DNA sequences encoding the consensus interferon can be synthesized as described in these patents, or other standard methods. The consensus interferon polypeptides are preferably expression products of engineered DNA sequences transformed or transfected into hosts, as described in this specification. That is, the consensus interferon is preferably obtained by the recombinant method. Such materials can be purified by methods well known in the art.

The above-described interferons and conditions suitable for therapy by gene replacement are for illustrative purposes and do not limit the scope of the present invention. Suitable interferon for the treatment of a known disease can be selected by a person skilled in the art without undue experimentation.

III. Methods for introducing polynucleotide sequences of secreted proteins into cells.

The term transformation or transform means any genetic modification of cells and includes both transfection and transduction.

As used herein, the term transfection of cells means the acquisition by a cell of new genetic material by introducing added DNA. Thus, transfection refers to the introduction of a nucleic acid (eg, DNA) into a cell using physical or chemical methods. Several methods of transfection are known to those skilled in the art, which include: DNA and calcium phosphate co-precipitation (Ms11yub8 Моη Mo1esi1ag Βίοίοβν. Uo1.7, SPSS TgaoPsg apb Exrge55yup Rgo1oso18, Eb. Eb. Miggau, Nitza Prg); the use of ΌΕΑΕ-dextran (see above); electroporation (see above); cationic transfection mediated by liposomes (see above); and microparticle bombardment, stimulated by tungsten particles (1oyp81op, 8.Α., Na1igs, 346: 776-777 (1990)); and co-precipitation of DNA and strontium phosphate (Vgazy EE s! a1., Mo1ss. Cs11. Vu1., 7: 2031-2034 (1987). All these methods are well known in this field.

In contrast, the term cell transduction refers to a technique for transferring a nucleic acid into a cell using viruses containing DNA or RNA. One or more isolated polynucleotide sequences encoding one or more interferon proteins contained in the virus can be included in the chromosome of the transduced cell. Alternatively, the cell is transduced with the virus, but this cell should not have an isolated polynucleotide in its chromosomes, and it should be able to express interferon in the cell outside the chromosomes.

In accordance with one embodiment of the invention, the cells transform (that is, genetically modify) ex y | yo. Cells are isolated from a mammalian organism (preferably a human) and transformed (i.e. transduced or transfected with ίη ugo) with a vector containing an isolated polynucleotide, in particular a recombinant gene, operably linked to one or more expression control sequences for expression of a recombinant secreted protein (e.g., interferon ) These cells are then administered to the recipient mammal to deliver the ίη 8Yi protein. The mammalian recipient is preferably a human and the cells to be modified are autogenic cells, i.e., cells isolated from a mammalian recipient. Methods of isolation and cultivation of ίη ugo cells are comprehensively described in the scientific literature.

In accordance with another embodiment of the invention, the cells are transformed or in any other way genetically modify модифициη у | уо. Mammalian recipient (preferably human) cells transform (i.e., transduce or transfect) ίη νί \ Ό with a vector containing an isolated polynucleotide, in particular a recombinant gene, operably linked to one or more expression control sequences to express a secreted protein (i.e., recombinant interferon ) with the subsequent delivery of this protein ίη 8Yi.

Isolated polynucleotides encoding a sectorized protein (e.g., cDNA encoding one or more therapeutic interferon proteins) are introduced into an ex νί \ Ό or ίη νί \ Ό cell by genetic transfer methods, such as transfection or transduction, to produce a genetically modified cell. Various expression vectors are known to those skilled in the art (i.e., vectors that facilitate the delivery of an isolated polynucleotide to a target cell).

The introduced genetic material usually includes an isolated polynucleotide, such as an interferon gene (usually in the form of cDNA including exons encoding interferon), together with a promoter that controls the transcription of the new gene. The specified promoter usually has a specific nucleotide sequence required to initiate transcription. Genetic material may optionally include introns that must be removed from the mature transcript by RNA splicing. The polyadenylation signal must be present at the 3'-terminal portion of the expressed gene. The introduced genetic material may also include an appropriate secretion signal sequence for secretion of the drug gene product (i.e., interferon) from the cell into the extracellular medium.

Optionally, the isolated genetic material may include additional sequences (i.e. enhancers) necessary to achieve the desired gene transcription activity. In accordance with the objectives, the enhancer is any untranslated DNA sequence that is associated with the coding sequence (in the cis position), with a change in the initial level of transcription determined by the promoter.

The isolated genetic material is preferably introduced into the cell genome immediately below the promoter, so that the promoter and the coding sequence are operably linked, resulting in transcription of the coding sequence. Preferred viral expression vectors include an exogenous promoter that controls transcription of the inserted interferon gene. Such exogenous promoters include both constitutive and inducible promoters.

Natural constitutive promoters control the expression of proteins that regulate the basic functions of cells. As a result, a gene controlled by the constitutive promoter is expressed under all cell growth conditions. Typical constitutive promoters are promoters for the following genes encoding specific constitutive or internal functions: hypoxanthine phosphoribosyltransferase (NRHT), dihydrofolate reductase (ΌΗΡΚ) (ΞοΙιαΓΓιηαηπ e! A1., Prg. -4630 (1991)), adenosine deaminase, phosphoglycerol kinase (CSC), pyruvate kinase, phosphoglycerolmutase, β-actin protomor (ba1 e! A1., Proc. No. 111. Asab. 8c1. I8A, 86: 10006-10010 (1989)) and other structural promoters known to those skilled in the art.

In addition, many viral promoters perform a constitutive function in eukaryotic cells. These promoters include: the early and late 8U40 promoters (see Vegηοίδΐ apb СΗатЬοπ. No. 11ige. 290: 304 (1981)); long terminal repeats (Ltn) of Molonei leukemia virus and other retroviruses (see. ); herpes simplex virus (H8U) thymidine kinase promoter (see ναβίκΓ e! a1., Proc. No. 11. Asab. δα. I8A, 78: 1441 (1981)); the earliest promoter of cytomegalovirus (1E1) (see Kagaziuata e! a1., 1. Exp. Meb., 169: 13 (1989); promoter of the Routh sarcoma virus (K.8U) (Υatato! ο e! a1., Ce11, 22: 787 (1980)); the main late promoter of adenovirus (Uataba e! A1., Proc. No. 11. Asab. Δα. I8A, 82: 3567 (1985)) and many others. Therefore, any of the above constitutive promoters can be used to control gene insertion transcription. (See also section B).

If an interferon gene is to be delivered to specific tissues, it is highly desirable to produce targeted expression of that gene. For example, in the scientific literature there are many promoters that are expressed only in certain tissues. Examples of such promoters are the liver-specific promoters of hepatitis B virus (LambC e! A1., Seme Tbegaru, 3: 1002-1009 (1996)) and the albumin gene (P ^ πke ^ (e! A1., Setez apb ^ eνе1οртеη !, 1: 268-276 (1987)); see also Sd e! A1., Sete Tbegaru, 3: 802-810 (1996) for another liver-specific promoter. In addition, many promoters are described in the scientific literature, which are expressed only in specific tumors, for example, the P8A promoter (prostate cancer), the carcinoembryonic antigen promoter (colon cancer and lung), the β-casein promoter (breast cancer), the tyrosinase promoter (melanoma), the calcineurin promoter Aa (glioma, neuroblastoma), the c-818 promoter (osteosarcoma) and the a-fetoprotein (hepatoma) promoter.

Genes controlled by inducible promoters are expressed only or mainly in the presence of an inducing agent (for example, transcription under the control of the metallothionein promoter is significantly enhanced in the presence of certain metal ions). See also glucocorticoid-inducible promoter present in the long terminal repeat of the mouse mammary tumor virus (MMTU LtN) (K1ez1e e! A1., S1. Ce11. Βίο1., 4: 1354 (1984)). Inducible promoters include reactive elements (NOT), which stimulate transcription when their inducible factors are linked. For example, there are reactive elements for serum factors, steroid hormones, retinoic acid, and cyclic AMP. You can select promoters containing a specific reactive element to obtain an inducible reaction, and in some cases, you can attach the reactive element to another promoter, which allows to report the inducibility of the recombinant gene.

Thus, choosing the appropriate promoter (constitutive or inducible; strong or weak), one can control both the presence and level of expression of interferon in a genetically modified cell. If the gene encoding interferon is controlled by an inducible promoter, interferon ίη zbi delivery starts when the genetically modified модифициη zbi cell is under conditions that allow transcription of interferon, for example, by injection of specific inducers of inducible promoters that control the transcription of this agent. For example, the expression of ίη zbi by genetically modified cells of the interferon protein encoded by the interferon gene under the control of the metallothionein promoter is enhanced by contacting ίη zbi of genetically modified cells with a solution containing the corresponding (i.e., inducible) metal ions.

Recently, very complex systems have been developed that allow precise regulation of gene expression by exogenously introduced small molecules. These systems include PK506 / rapamycin system (VAaega! tetracycline system (Sozzeη! A1., Borrow, 268: 1766-1768, 1995); ecdysone system (еο e! a1., Pr. -243, 1997).

Thus, the amount of interferon delivered by ίη zbi is regulated by controlling factors such as (1) the type of promoter used to directly transcribe the introduced gene (i.e. whether the promoter is constitutive or inducible, strong or weak, or tissue-specific);

(2) the number of copies of an exogenous gene introduced into the cell; (3) the number of transduced / transfected cells introduced (e.g., implanted) into the patient; (4) the size of the implant (for example, an implant or an encapsulated expression system) when implementing ex νίνο methods; (5) the number of implants in the implementation of ex νίνο methods; (6) the number of transduced / transfected cells with the introduction of ίη νίνο; (7) the duration of transduced / transfected cells or implants in the body during the implementation of the ex νίνο and ίη νίνο methods; and (8) the rate of production of interferon by a genetically modified cell.

Those skilled in the art can select and optimize these factors to deliver a therapeutically effective dose of a particular interferon without undue experimentation, taking into account the above factors and the patient’s clinical chart.

Since the protein expressed in accordance with our hemotherapy methods is a secreted protein, the surrounding cells that do not contain the gene therapy vector are still exposed (see examples). Based on this, the methods of the present invention usually do not require the use of a selective gene. However, in addition to at least one promoter and at least one isolated polynucleotide encoding interferon, the expression vector may optionally include a selective gene, for example a neomycin resistance gene, to facilitate the selection of cells that have been transfected or transduced with expressing by vector. Alternatively, cells are transfected with two or more expression vectors, of which at least one vector contains the gene (s) encoding interferon (s) and the other vector contains a selective gene. One of skill in the art can easily select the desired promoter, enhancer, selective gene and / or signal sequence (described below) without undue experimentation.

IV. Methods for producing specific vectors for gene therapy.

Any methods known in the art for inserting polynucleotide sequences into a vector can be used. See, for example, ΞαιηόΐΌοΚ e! a1., ΜοΕοιιΙαΓ Οοηίη§: Α Εαόοга South of Mayia1, C. О16 Ϊ́άοΐά 8ртшд Ηа ^ Ьο ^, ΝΥ (1989) and Аи8иее1 е! A1., Sitgesh Ρτοΐοοοίδ ίη ΜοΕοιι1; · ιγ Βίοίο ^, 1. \ UPeu & δοηβ, ΝΥ (1992). Typical vectors include acceptable transcriptional / translational control signals that are operably linked to the polynucleotide sequence of a particular interferon. Promoters / enhancers can also be used to control the expression of interferons. (See section III).

Expression vectors compatible with mammalian host cells for gene therapy of tumor cells include, for example, plasmids; retroviral vectors of birds, mice and humans; adenoviral vectors; herpes virus vectors; parvoviruses and non-replicative poxviruses. In particular, recombinant replication-defective viruses can be obtained in packaging cell lines that produce only replication-defective viruses. See Stylet Ρτοΐοοοίδ ίη Μο ^ ου ^ Βίοίο ^: 8сс1юп8 9.10-9.14 (АиццЬе1 е! А1., Ебз.). Sgien RNYmshsch Α88οс ^ а! Е8, 1989.

Specific viral vectors for use in gene transfer systems are well known. See, for example: Mab / ak e! A1., 1. Sep. νίτοί., 73: 1533-36 (1992) (papovavirus 8M40); Branch e! A1., Sigg. Τορ. M1go1. Ittii 1., 158: 39-61 (1992) (adenovirus); Μο§8 e! A1., Sigg. Τορ. McGoyuk Ittiyo1., 158: 25-38 (1992) (vaccinia virus); MiζλΌζΐΜΤ Sigg. Τορ. Muguguk Ittii1, 158: 97123 (1992) (adeno-associated virus); Magdickey, Sigg. Τορ. McGück Ittiyo1., 158: 6793 (1992) (herpes simplex virus (Ηδν) and Epstein-Barr virus (ΗΒν)); M111eg, Sigg. Τορ. M1GGOB1. Ittii 1., 158: 1-24 (1992) (retrovirus); Β ^ aibuορabyuau e! A1., Μο1. Ce11. Βίο1., 4: 749-754 (1984) (retrovirus); M111eg e! A1., No! IgE, 357: 455-450 (1992) (retrovirus); ^ι ^ ι ^ οη, Loan 256: 808-813 (1992) (retrovirus).

Preferred vectors are DNA-containing viruses that include adenoviruses (preferably Ab-2 or Ab-5 based vectors), herpes viruses (preferably herpes simplex vectors) and parvoviruses (preferably defective or non-autonomous parvovirus vectors, more preferably vectors based on adeno-associated virus and most preferably vectors based on ΑΑν2). See, for example, Αΐί e! A1., Seme Τye ^ aρу, 1: 367-384, 1994; US patent No. 4797368 and 5399346 and the following descriptions.

The choice of a particular vector system, for example, for transferring an interferon sequence, depends on a number of factors. One important factor is the type of target cell population. Although retroviral vectors have been extensively studied and used in a number of gene therapy applications, they are usually not suitable for infection of cells that do not divide, but may be useful in the treatment of malignant tumors, since they integrate and express their genes only in replicating cells. They are applicable in ex νίνο methods and attractive in this regard due to the stable integration of the target cells into the genome.

Adenoviruses are eukaryotic

DNA viruses that can be modified to efficiently deliver a drug or reporter trans21 gene to different types of cells. Methods are currently being developed for using conventional types 2 and 5 adenoviruses (Ab2 and Ab5, respectively), which cause human respiratory disease, for gene therapy of Duchenne pseudohypertrophic myopathy (ΌΜΌ) and cystic fibrosis (SR). Ab2 and Ab5 belong to a subclass of adenoviruses that are not associated with human cancers. Adenoviral vectors are capable of providing exceptionally high degrees of transgene delivery to virtually all cell types, regardless of mitotic state. High titers (10 ¹¹ plaque-forming units / ml) of the recombinant virus can be easily obtained in 293 cells (line transformed with adenovirus, complementary human kidney embryonic cells: ATCC SK151573), and they can be stored in a cryostat for long periods of time without significant losses. The effectiveness of this system with respect to the delivery of the therapeutic transgene ίη νίνο, balancing the genetic imbalance, has been demonstrated in animal models of various diseases. See Υ. GSaapa bc, A1yego8s1ego818, 36: 261-268 (1986); K. Ταηζα \ να е! A1., REV8 Heyege, 118 (1): 81-84 (1980); Oo1a8! En e! A1., Hei 1. Meb., 309 (11983): 288-296 (1983); 8. 1 ₈ Lb8e e! A1., 1. C1sh. Ιηνοδί., 92: 883-893 (1993); and 8. 18THE8Y e! A1., 1. C1sh. 1 # 8!., 93: 1889-1893 (1994). A recombinant replication-defective adenovirus encoding cDNA for the transmembrane cystic fibrosis regulator (CPTC) has indeed been used in several clinical trials involving cystic fibrosis patients. See, for example, 1. ^ ί18οη, Νι! Ige, 365: 691-692 (October 21, 1993). Further confirmation of the safety of recombinant adenoviruses for gene therapy should be the large-scale testing of live vaccines from adenovirus for vaccination of the population.

Human adenoviruses include a 36-strand linear double-stranded DNA genome. n., which is divided into 100 units of the genetic map, the length of each of which is 360 bp. DNA contains short inverted terminal repeats (ΙΤΡ) from each end of the genome, which are necessary for viral DNA replication. Gene products are located in the early (E1 to E4) and late (L1 to L5) regions based on expression before or after the initiation of viral DNA synthesis. See, for example, Nail, U1to1odu, 2b eb., Eb. B.K. P1e1b8, Ratep Rge88 b! B., No. \ ν Υο ^ k (1990).

The adenovirus genome carries out a strictly regulated program throughout its normal life cycle. See Υ. Υηηβ e! A1., Proc. No. I1. Asab. 8s1. I8A, 91: 44074411 (1994). Virions are internalized by cells and enter the endosome, from where the virus enters the cytoplasm and begins to lose its protein coat. Virion DNA migrates to the nucleus, where it retains its extrachromosomal linear structure, and does not integrate into the chromosome. The earliest genes, E1a and E1b, are expressed in the nucleus. These early gene products regulate the transcription of adenovirus, which is why they are necessary for the replication of the virus and expression of a number of host genes (which serve as the seed forcing the cell to produce the virus) and are central to the cascading activation of delayed early genes (for example, E2, E3 and E4). followed by later genes (e.g. E1-L5).

The recombinant adenoviruses with a replication defect of the first generation, designed for gene therapy, have deletions of the entire E1 a and part of the E1b regions. This replication-defective virus is grown in 293 cells, which contain the adenovirus functional region E1, which provides trans-E1 proteins, which makes it possible to replicate adenovirus with an E1 deletion. The resulting virus is able to infect many types of cells and can express the introduced gene (provided that it carries a promoter), but cannot replicate in a cell that does not carry the DNA of the E1 region. The advantage of recombinant adenoviruses is that they have many different hosts, can infect dormant or differentiated cells in the final region, such as neurons, and are practically non-oncogenic. Adenoviruses do not appear to integrate into the host genome. Since they exist outside the chromosome, the risk of insertional mutagenesis is significantly reduced. Recombinant adenoviruses produce very high titers, while the viral particles are moderately stable, high levels of expression are achieved and thus a large number of cells can be infected.

Adeno-associated viruses (AAUs) have also been used as vectors for somatic gene therapy. AAU is a small virus containing single-stranded DNA, with a simple genomic organization (4.7 kb), which makes it an ideal substrate for recombinant DNA technology. Two open reading frames encode a series of her and sar polypeptides. The re-polypeptides (her78, her68, her62 and her40) are involved in the replication, conservation and integration of the AaU genome. Sar proteins (UR1, UR2 and UR3) form a capsid of the virion. The open reading frames of the ger and sar polypeptides in the 5'- and 3'-terminal regions are flanked by inverted terminal repeats of 145 bp in length. (ΙΤΚ.), Of which the first 125 bp capable of forming Υ- or T-shaped duplex structures. Of great importance for the construction of AAU vectors is that all her- and sardomens can be excised and replaced with a drug or reporter transgene. See RE. Saisg. ίη б Нап ок о о о Г ату Ratuouiikek, eb., R. Τί_) 88eg, SHS Rgekk, pp. 155-168 (1990). It was found that inverted terminal repeats represent the minimum sequence required for replication, conservation, packaging and integration of the ALU genome.

AAU has a two-phase life cycle consisting of latent and lytic episodes. During latent infection, AAU virions enter the cell in the form of encapsulated single-stranded DNA and are soon transferred to the nucleus, where AAU DNA stably integrates into the host chromosome without requiring division of the host cell. In the absence of a helper virus, the integrated AAU genome remains latent, but it can be activated and saved. The lytic phase of the life cycle begins when a cell carrying the AAU provirus is reinfected with the herpes virus or adenovirus encoding the functions of the helper virus that AAU needs to excise from the host chromatin (VD Site, see above). Parental single-stranded DNA infection spreads to duplex replicative form (BP) DNA in a ger-dependent manner. The stored AAU genomes are packaged in preformed protein capsids (twenty-sided symmetry with a diameter of approximately 20 nm) and are isolated as infectious virions that contain packed + or β-genomes of single-stranded DNA after cell lysis.

The use of AAU is associated with great prospects in gene therapy. Viral particles are very stable and recombinant AAUs (GAAUs) have good drug-like characteristics, expressed in that GAAUs can be purified by precipitation by centrifugation or by delamination with a Ccc1 gradient. They are heat-resistant, can be lyophilized to form a powder and rehydrated while maintaining full activity. Their DNA is stably integrated into the host chromosomes; therefore, expression is long-term. They have a wide host range, and AAA does not cause known diseases, so the recombinant vectors are not toxic.

It has been established that high gene expression from AAU in mice occurs for at least 1.5 years. Cm. Since there is no evidence of viral toxicity or immune response of the host cell, it can be concluded that these limitations related to viral gene therapy are currently overcome.

Karyy, Leope, 8at1kk1, X1ao, RGaGG, O'Ma11eu apb Etude (1994), No. 11ige Oepeysk, 8,

148-153, described prolonged (up to 4 months) expression of tyrosine hydroxylase in rat brain after direct intracranial injection using the AAU vector. This method can be very promising for the treatment of Parkinson's disease in humans. Expression was very effective and the virus was safe and stable.

E1kiet e! A1., (No. 1 igu Mebushe, (1997) 3, 306312) reported stable gene expression in mice after intramuscular injection of AAU. In this case, the virus was also safe. No cellular or humoral immune response was detected against this virus or foreign gene product.

Kek81et e! A1., (Proc. No. 111. Asab. δει. I8A, (1996) 93, 14082-14087) reported high expression of the erythropoietin gene (EPO) after intramuscular injection of AAU in mice. It was found that the EPO protein is present in the bloodstream, while an increase in red blood cells was noted, which indicates the therapeutic potential of this method. In another study, performed by the same team of researchers, AAU expressing the gene for H8U was used to treat malignant tumors. High gene expression was detected in solid tumors.

Recently, it has been demonstrated that a recombinant baculovirus isolated from AI! Odgarya saigottsa baculovirus, which is a multiple nuclear polyhedrosis virus (AsMIRU), is capable of transducing mammalian cells. (See NoGtapp, S., 8apb1d, U., 1epshpdk, O., Vibo1ry, M., Zsy1ad, R. apb Zyaykk, M. (1995), Egyshep! Depe papGeg w! O yitap yer! , Proposal, Ass. 8sk from A, 92, 1009910103; Vouse, R.M. apb Visyeg, K.B.V. (1996) Vasi1ou1ti8-teb1a1eb depec yapkGeg t! O tattayap se11k, Proc. 93, 2348-2352).

Recombinant baculovirus has several important benefits for gene therapy. Such advantages include the ability to insert very large DNA, the absence of a previous immune response in humans, the absence of replication in mammals, the absence of toxicity in mammals, the lack of expression of viral genes in mammalian cells due to the insect specificity of the transcriptional promoters of baculovirus and probably the absence of the cytotoxic T reaction lymphocytes directed against these viral proteins.

U. Test for efficacy and identification of interferons.

Interferon polynucleotides are introduced into the cell using an expression vector.

Typically, the effectiveness of this vector for gene therapy in a certain state of the cell and metabolism are tested by analyzing: (ί) changes in cell morphology; (ίί) suppression of cell proliferation; and (w) antiviral effect.

In order to select and optimize a particular expression vector for expressing a specific interferon gene product in an isolated cell, an interferon gene is prepared, preferably having one or more suitable control regions (eg, a promoter); create a vector construct containing a vector with the insertion of the interferon gene; transfected or transduced cultured ίη νίίτο cells with this vector construct; and determine the presence of an interferon product in cultured cells.

The result of transfection with polynucleotides encoding interferons can be verified ίη νίίτο using any available human tumor cell line. These cells include human bladder cancer cell lines, 5637 (ATCC NTV9), human breast cancer cell lines, ΜΌΑ-ΜΒ-468 (ATCC NTV 132); human prostate cancer cell line, 145υ 145 (ATCC NTV81); human osteosarcoma cell line, 8–82 (ATCC NTV85); fibrosarcoma cell line with metastases that transform into a malignant tumor of a human lung, Ηκ913Τ (ATCC ΗΤΒ152); cell line of a malignant tumor of the human cervix, Heb (ATCC EC2). All these cell lines can be transfected with the corresponding polynucleotides encoding interferon, and the result of transfection on the growth and morphology of the cells can be tested using methods known in this field, such as the method of eliminating trypan blue to measure cell viability, counting cells to measure their reproduction with over time and the incorporation of tritiated thymidine to measure DNA replication.

The effect of secreted protein on surrounding cells that do not contain a viral vector with the corresponding polynucleotides encoding the protein can be easily tested using the methods described in example 3.

Once introduced into the target cell, interferon sequences can be identified by conventional methods, such as nucleic acid hybridization using probes containing sequences that are homologous / complementary to the inserted mutant interferon sequences. Interferon transcription can be measured by polymerase chain reaction using reverse transcriptase. Alternatively, the interferon protein is measured in a cell-conditioned medium using a conventional antiviral assay or enzyme-linked immunosorbent assay (E18A). According to another approach, one or more sequences can be identified by the presence or absence of marker gene function (e.g., thymidine kinase activity, antibiotic resistance, and the like) caused by the introduction of an expression vector into the target cell. For example, if a polynucleotide encoding beta-interferon 1a is inserted into a vector having a dominant selective marker gene, such as a neomycin phosphotransferase gene, separately controlled by the early 8U40 promoter, the sequence can be identified by the presence of marker gene function (resistance to geneticin). Other methods for detecting the corresponding vector are also well known to those skilled in the art.

VI. Utility.

A. Interferons and infectious diseases.

Interferons are used to treat bacterial, fungal and viral infections. Influenza and vesicular stomatitis viruses (νδν) are especially sensitive to suppression by interferons and are often used in analyzes to determine the activity of interferon and in studies performed on the basis of the mechanism of antiviral activity of interferon. Other viruses that are human pathogens and appear to be sensitive to interferons include hepatitis B virus (ΗΒν), hepatitis C virus (ΗΟν), hepatitis вирус virus, human papilloma virus, herpes simplex virus, herpes zoster virus, cytomegalovirus ( ί, 'Μν). rhinitis virus and encephalomyocarditis virus.

Hepatitis is one of the most promising diseases for gene therapy. Viral hepatitis, that is, liver disease caused by many viruses, is a major public health problem worldwide. Five different human hepatitis viruses are currently isolated and cloned. These are hepatitis A, B, C, Ό, and E. viruses. Some cases of acute and chronic viral hepatitis appear to be associated with still little known hepatitis viruses, such as the recently discovered hepatitis C virus. The most widespread viruses are A, B, and C. In addition to acute and chronic damage and inflammation of the liver, infectious diseases caused by иν and ΗCV viruses can lead to malignant hepatoma. Interferons have shown a rather effective effect of ίη νΐνο against ΗΒν and ΗCV, as well as against hepatitis Ό and hepatitis A in cell culture.

Viral hepatitis can be treated by introducing the interferon gene. Preferred cells for introducing this gene are hepatocytes in the liver. Although an ex ν можноνο gene therapy method can be used in this case (for example, by explanting liver cells, introducing a polynucleotide expressing interferon, and then transplanting these cells to the patient), the доставкаη νίνο gene is the preferred method. The gene is injected into the portal vein of the liver by surgery or inserted through a catheter into the hepatic artery. A less invasive technique such as intravenous injection is widely used.

The interferon gene is controlled by a transcriptional promoter in an acceptable expression vector. The transcriptional promoter in nature can be cellular or viral (SMU, 8U40, P8U, etc.). To achieve specific gene expression in the liver, a hepatocyte-specific cell promoter, such as an enhancer / albumin promoter, an A1-antitrypsin promoter or an HBV enhancer / promoter, is preferably used. The scientific literature describes many liver-specific promoters (see above).

In addition, you can construct a vector in which the interferon gene is expressed only in cells infected with hepatitis. For example, expression of an interferon gene can be induced by the HBx transcription factor of HBV, which is present only in HBV-infected hepatocytes. In addition, a carrier system can be used. The delivery system may be non-viral; for this purpose, cationic liposomes or protein conjugates can be used: DNA (DNA conjugates with azialoglycoproteins can be delivered to the liver by binding to the azialoglycoprotein receptor on hepatocytes).

However, currently the most efficient gene delivery systems are of viral origin. Recombinant retroviruses are used to deliver genes to human hepatocytes ex νίνο. Animal models show that recombinant adenoviruses can very effectively localize in the liver after intravenous administration of ίη νίνο. Approximately 98% of adenoviruses administered intravenously are localized in the liver.

Obviously, recombinant adeno-associated virus (GAAU) can also infect the liver after the introduction of ίη νίνο. Other types of viruses can be used, such as alpha viruses, lentiviruses, or other mammalian viruses. It has recently been discovered that a non-mammalian virus, in particular an insect-specific baculovirus, is able to efficiently deliver and express genes in hepatocytes (ΗοίΊηαη e1 a1., 1995; Wous apb VisBeg, 1996, see above).

For example, recombinant adenoviruses can be used to deliver the interferon gene ίη νίνο. Replication-defective adenoviruses were constructed by several research teams. The first generation of such viruses is defective due to deletion of the E1 region. This defect complements in cell line 293, which expresses the E1 region of the adenovirus. It is desirable to use a recombinant adenovirus, which is more damaged by a deletion of the E1, E2a and / or E4 genes. All of these deleted functions can be expressed in the packaging cell line. The interferon gene is introduced downstream of any large number of promoters (for example, the earliest SMU promoter, P8U long terminal repeat, cell actin promoter, albumin enhancer / promoter, and other liver specific promoter). This gene cluster is introduced into a vector based on recombinant adenovirus instead of one of the deleted genes, such as E1, in order to create an adenovirus transfer vector.

Recombinant adenoviruses having the interferon gene are obtained by direct ligation or homologous recombination after transfection of packaging cells by standard methods. The strain of the recombinant virus having the interferon gene is subjected to hemolytic purification several times and then produced in large volume in packaging cells. The virus can be cleaned by stratification of C'ASH column chromatography or other methods and then titrated on packaging cells. The scientific literature describes in detail the methods for obtaining adenoviruses with a defect in E1 and E2a (EpdeHagb! E1 a1., Proc. No. 11. Asab. 8c1. I8A, 91: 6196-6200, 1994) and adenoviruses with a defect in E1 and E4 (Syu e1 a1., ί. νίΐΌΐοβν, 70: 8934-8943, 1996). Methods for the preparation of adenoviruses with a defect in E1 are presented in the work of Chabat, G. but about b. Rgeues, Me11yub5 £ ογ Sop51gis1yup οί Lbepou | gi5 UesGogh, Mo1. Vyues11., 3: 207-220 (1995)).

Different doses of viruses should be tested. The dose of the virus, apparently, ranges from 10 ⁷ plaque forming units (PFU) to 10 ¹² PFU. If necessary, the virus can be re-introduced (for example, once a month for 1-6 months). The humoral immune response resulting from repeated administration of the virus may limit the effectiveness of repeated administration. In this case, an immunosuppressant, such as cyclosporin or an antibody, against the CE40 ligand can be introduced together with the virus.

The effect of gene therapy with interferon against viral hepatitis can be tested in animal models (see section C). For example, there are many animal models used to test the hepatitis B virus. These animals are marmots, ducks, blunts, rats and mice. As animal models of IEDs, mice that have a transgene for IEDs and stably replicate can be used.

IVV DNA in the liver, which leads to the constant production of the virus IVA in the bloodstream. In the study of NSOs, chimpanzees can be used as an animal model. An adenovirus having the interferon gene can be injected directly into the liver or as an intravenous injection. To determine the effectiveness of treatment, they control the replication of viral DNA by viral particles in the bloodstream, liver enzymes (LTE), pathology and inflammation of the liver.

B. Malignant tumors.

It has been established that interferon proteins have an anti-oncogenic effect in the case of many malignant tumors. For an introduction to research in this area, see ED-61et aib 8ο1ι \ ναΓίζ. Sense g Vekeatsy, 50: 34733486, 1990; Mapt-Obedagb, ΌΝ & Ρ, 4: 116-117, 1991; and 8p1dee1, Zestagk ίη Οικοίοβγ, 15 (5): 4145, 1988. Treatment with alpha interferon and betainterferon has proven effective in several types of malignant tumors. Gene therapy can be carried out alone or in combination with conventional methods such as surgery, radiation therapy or chemotherapy. The following list of malignant tumors to be treated with gene therapy is very incomplete, and it is likely that interferon gene therapy can give positive results in a number of malignant tumors that are not included in this list.

Malignant gliomas account for 6080% of all primary brain tumors in adults. Human glioma cells can be implanted intracerebrally in immunocompromised mouse mice to obtain a glioma model. It was found that treatment with betainterferon protein increases the survival of these mice. In trials for the treatment of gliomas with betainterferon, there is a problem of high toxicity caused by parenteral administration (intravenous or intramuscular) of beta-interferon. Localized delivery of the beta-interferon gene to the brain, for example, during surgery, can provide long-term production of beta-interferon in the brain without the occurrence of side effects that occur after systemic administration of the protein.

Melanoma is a very promising disease for interferon gene therapy. The prognosis of metastatic malignant melanoma is poor. The incidence of melanoma is constantly increasing and its treatment with conventional chemotherapy methods is ineffective. Melanoma appears to be an immunogenic tumor, so the patient’s response may depend on the host’s immune response. In this case, both antiproliferative and immunomodulatory effects of beta-interferon can give an effective result. We were convinced that the beta-interferon protein has a direct antiproliferative effect on cultured malignant melanoma cells.

Hemangioma is the proliferation of capillary endothelium, leading to the deposition of mast cells, fibroblasts and macrophages and ultimately causing tissue damage. Although hemangiomas are usually harmless, they can affect vital organs and cause death. It has been established that the alpha interferon protein causes the reverse development of steroid-resistant hemangiomas at an early stage of development in young children (Mayt-Obedagb, see above).

Interferon proteins, as you know, can effectively treat leukemia, lymphoma and myeloma. This result contradicts the general opinion that, despite the high efficiency of interferon proteins for the treatment of ίη νίίτο cancer, they are much less effective in treating приη νινο. And yet, as shown by clinical trials involving humans, alpha-interferon can effectively treat malignant reticuloendotheliosis, chronic myelogenous leukemia, skin cell lymphomas, Hodgkin's lymphoma and multiple myeloma. Betainterferon protein inhibits the growth of kidney cancer cells in culture. ΙΕΝ-α is already certified for use in the treatment of kidney cancer.

A malignant tumor of the colon and rectum is the main cause of mortality in cancer patients in the United States. Proteins ΙΕΝ-α, ΙΕΝ-β and ΙΕΝ-γ exert a strong antiproliferative effect on cultured human colon cancer cells. A malignant tumor of the colon often gives metastases to the liver with life-threatening consequences. An adenovirus or other liver-specific delivery systems can be used to deliver the interferon gene to the liver to target these metastases. Treatment for malignant hepatoma can be very successful due to the high efficiency of delivery to the liver provided by adenovirus. The-β protein was found to significantly inhibit the proliferation of human hepatoma cells in culture.

Interferon proteins can effectively treat inoperable non-small cell lung cancer in some clinical trials and do not give such a result in other trials. It was established that two cell lines of a malignant tumor of a human lung are sensitive to growth inhibition by beta-interferon protein (beta-interferon was more effective than alpha-interferon). In one clinical trial, after intravenous administration of interferon, significant interferon-related toxicity was observed. Local delivery of the beta interferon gene to the lung (for example, aerosol administration of the recombinant adenovirus vector) can give good results without causing the toxicity observed in the case of systemic delivery of this protein. When performing ίη νίίτο studies, betainterferon protein inhibited cell proliferation of small cell lung cancer.

The alpha interferon protein inhibits the growth of xenografts of malignant breast tumors in mice peibe. Betainterferon can give effective results in the treatment of a malignant breast tumor, not only due to its antiproliferative effect, but also due to the induction of estrogen and progesterone receptors ίη νίνο, which results in sensitization of the malignant breast tumor to tamoxifen of the antiestrogen. The following are experimental data on the use of ΙΕΝ-β gene therapy for the treatment of human breast cancer in mice used as animal models (see examples 3 and 4).

A malignant ovarian tumor can also be treated with this method. The betainterferon protein appears to be less active than alpha interferon in inhibiting the proliferation of cultured ovarian cancer cells. In this case, the vector used in gene therapy is injected into the peritoneum, since a tumor of this type tends to fill the abdominal cavity.

In short, interferon proteins exhibit anti-oncogenic properties in a number of diseases, although clinical trials using the interferon protein are not universally positive. Proteins ΙΕΝ-α and ΙΕΝ-β were tested in combination with conventional chemotherapy methods, and it turned out that in many cases their effect is synergistic with these drugs, including cells of a malignant tumor of the cervix uteri, malignant tumor of the larynx, leukemia, malignant tumor of the kidney, adenocarcinoma of the thick intestines and myelomas. In addition, it is believed that interferons have activity against angiogenesis. There is an inverse correlation between local ΙΕΝ-β levels and angiogenic ability. Some data show that in order to suppress angiogenesis, it is necessary to maintain the concentration of ΙΕΝ-β at a high level. In this case, gene therapy with interferon should be preferable to a protein treatment in which a high interferon content may suddenly drop or not be detected at all.

C. Animal models for gene therapy.

Specialists in this field should be aware of many animal models used to test the gene therapy methods ex νίνο and известныη νίνο. The most common animal model of a malignant tumor are rodents, in particular nude mice (pi / pi) with a xenograft of a human tumor. Human malignant tumor cells are propagated in culture, after which they are transfected or infected with an interferon-encoding gene operably linked to appropriate expression control sequences. These cells are then injected into nude mice. Tumor cells are usually injected subcutaneously into the back of the mouse, which leads to the formation of a solid tumor (see example 1). Alternatively, tumor cells can be injected orthotopically into the organ where they usually develop (lung cancer cells are injected into the lung; colon cancer cells are introduced into the colon, etc.). Tumor growth is monitored by measuring the diameter of the tumor over a period of time (see Example 2).

Okaba e1 a1., (1996), Seme Tiegaru, 3, 957964, obtained experimental gliomas in mice by direct intracranial stereotactic injection of human glioma cells into the brain. After the formation of detectable tumors, the AAU vector expressing the herpes simplex virus thymidine kinase gene (1 k H8U) was introduced into the same area as a direct injection. The 1k H8U enzyme converts ganciclovir (SSV) of the non-toxic nucleoside analog into a toxic metabolite. After gene therapy, SSA was administered intraperitoneally. In mice that were injected with AAULK and SSU, rather than control AAU or AAU-1k without SSU, a significant decrease in tumor size was observed. This treatment method is safe and effective.

Recombinant adenoviruses (ABs) have also been used to treat solid tumors in animal models and in initial clinical trials in humans. Many of these studies used similar nude mouse / human xenograft models. Below are some examples of these modeling experiments, C1auten e1 a1., (1995), Sansa g Kekeagsy, 55, 1-6, created a model of squamous malignant tumor of the scalp and neck of mouse mice. They found that adenovirus expressing wild-type p53 interferes with the formation of these tumors.

Н | Г5с1ю \\ 'Цх е1 а1., (1995), Nitaη Sene

Tiegaru, 6, 1055-1063, introduced cells of a colon cancer of a human colon into mice. After the formation of tumors, they injected directly into these tumors adenovirus 33 expressing the E.co11 cytosine deaminase (CO) gene, and then 5-fluorocytosine (5ES) was systemically injected (CO and 5EC are an enzyme / prodrug combination that is similar to 1k and OSU). These researchers observed a 4-5-fold decrease in tumor size.

ΖΙππίβ e! A1., (1996), Proc. No. And. Asab. 8sk IBL, 93 4513-4518, received human mammary gland tumors in mice. Adenovirus expressing alpha interferon was injected directly into these tumors. These researchers observed a 100% tumor reduction in these animals.

Ko e! A1., (1996), Nitap Sepe Tkegaru, 1, 1683-1691, received human prostate tumors from mice Pibe, and found that direct intratumoral injection of wild-type adenovirus expressing p53 inhibits tumor growth. All injected mice had no tumor for at least 12 weeks after treatment.

VBSKOGG e! A1., (1996), 8aepse, 274, 373-376, received cervical and glioblastoma tumors in Pibe animals. They introduced adenovirus with deletion of the E1B gene to these mice. In the absence of E1B, adenovirus selectively destroys tumor cells with p53 deficiency. When injected directly into the tumor, this adenovirus caused tumor reduction in 60% of the animals.

Ok ^ aba e! A1., (1996), Nitap Sepe Tkegaru, 7, 1567-1576, injected human colon cancer cells into the liver of mice peba to mimic the metastases of a colon cancer in the liver. Then, adenovirus expressing CO was injected into the liver near the tumor to these mice. Systemic administration of 5EC inhibited tumor growth in these animals.

Models of malignant tumors can also be created in immunocompetent mice and rats. These tumors can be obtained from tumor cells of syngeneic rodents injected into mice. Alternatively, these tumors can be obtained from endogenous cells. In these cases, endogenous tumors may result from the introduction of a carcinogen to the animal, or alternatively they may spontaneously form due to the genetic environment of the mouse (for example, due to p53 deficiency).

Ekkat1 e! A1., (1996), Nitap Sepe Tkegaru, 7, 141-148, treated endogenous mesothelioma in immunocompetent rats with adenovirus expressing the 1k gene. This virus was injected into the pleural cavity, after which SSV was systemically administered. These researchers observed a significant decrease in tumor mass and an increase in survival time.

Eikat e! A1., (1996), Nitap Sepe Tkegaru, 7, 515-523, implanted prostate tumor cell lines of syngenic mouse subcutaneously to immunocompetent mice. Then they injected adenovirusbk directly into the tumor and systemically injected SSRs. The authors reported a decrease in tumor size and an increase in the life span of mice.

Whackup e! A1., (1996), Nitap Sepe Tkegaru, 7, 1995-2002, injected an adenovirus expressing the cytokine BB-12 directly into breast tumors of endogenous mice. They found that in 75% of the mice, the tumors decreased and in the remaining 33% of the mice, the tumors were completely absent for an extended period of time.

Kbeu e! A1., (1996), No. 11, Mebkte, 2, 13361341, injected an adenovirus expressing the retinoblastoma gene directly into melanotrophic pituitary tumors that arose spontaneously in K + + mice. These researchers found a decrease in tumor cell proliferation, a decrease in tumor growth, and an increase in life expectancy in experimental animals.

In clinical studies of human gene therapy, retroviral vectors were the first to be used. Report Ko1k e! A1., (1996), No. 11, Mebkte, 2, 985-991, is directly related to this patent application. This report describes a recombinant retrovirus expressing a wild-type p53 gene. This virus was introduced to nine patients suffering from non-small cell lung cancer, as a direct intratumoral injection using a needle inserted into a bronchoscope. Of the nine patients, three showed reverse tumor development, and another three patients showed stabilization of tumor growth.

Ό. Other embodiments of the invention.

Genetically modified cells are injected, for example, by intraperitoneal injection, or they implant cells, a graft or a capsule containing cells into a portion of the recipient's body that is compatible with these cells. As used herein, the term “cell compatible region” means a recipient structure, cavity, or fluid into which a genetically modified cell (s), a cell transplant, or an encapsulated cell expression system can be implanted without any harmful physiological consequences. Typical cell-compatible areas are, for example, the peritoneal, pleural and pericardial cavities, as well as the solid tumor from which these cells were isolated, or the organ from which the tumor was removed.

These genetically modified cells are implanted in a cell-compatible region separately or together with other genetically modified cells. Thus, it is within the scope of the present invention to modify a recipient system using a mixture of genetically modified cells, such that the first modified cell expresses the first interferon and the second modified cell expresses the second interferon or other secreted protein. Other types of genetically modified cells (e.g., hepatocytes, smooth muscle cells, fibroblasts, glial cells, endothelial cells or keratinocytes) can be introduced together with genetically modified cells to produce expression of a complex set of introduced genes. In addition, several recombinant genes on the same or different vectors can be introduced into each genetically modified cell, resulting in the expression of several interferons in one cell.

The present invention further relates to a cell transplant. This graft includes several of the above genetically modified cells attached to a carrier suitable for implantation into a recipient mammal. The carrier can be obtained from natural or synthetic material. In accordance with another embodiment of the invention, such a graft contains a piece of peritoneum. According to this embodiment of the invention, the carrier is a natural matrix holding together a plurality of genetically modified cells. Alternatively, this graft contains many of the cells described above attached to a substitute for a natural matrix (for example, gel foam (irrigation, Ka1atahoo. Μίοΐι.). Dacron, Cortex®).

Another object of this invention is an encapsulated cell expression system. The encapsulated system includes a capsule suitable for implantation into a recipient mammal and a plurality of genetically modified mesothelial cells described above. This capsule can be made from synthetic or natural material. Methods for making such capsules are well known to those skilled in the art. Unlike cells that are implanted directly into the body of a mammalian recipient (that is, implanted in such a way that genetically modified cells are in direct physical contact with a site compatible with the cells), the encapsulated cells remain isolated (that is, are not in direct physical contact with the focus of the disease ) after implantation. Thus, the encapsulated system is not limited to a capsule containing genetically modified non-immortalized cells; it can also include genetically modified immortalized cells.

VII. Medications.

According to a preferred embodiment of the invention, the preparation of genetically modified cells contains a sufficient number of cells to deliver a therapeutically effective dose of interferon to a recipient ίη δίΐιι. One of skill in the art can determine the therapeutically effective dose of a particular interferon for a given disease without undue experimentation. So, to determine the effective dose, a specialist in this field must take into account the patient’s condition, the severity of the disease, as well as the results of clinical studies of a particular interferon intended for administration.

If the gene or genetically modified cells have not been previously introduced in a pharmaceutically acceptable carrier, they are placed in such a carrier immediately before administration to the recipient. Such pharmaceutically acceptable carriers include, for example, isotonic saline and other buffers that are suitable for administration to a patient and for a given treatment method.

The term pharmaceutically acceptable carrier means one or more ingredients, natural or synthetic, with which the isolated polynucleotide encoding interferon is mixed to facilitate its administration to a needy subject. An acceptable carrier is sterile saline, although other aqueous and non-aqueous isotonic sterile solutions and suspensions should be known to those skilled in the art. In this regard, the term “carrier” means any plasmid and expression viral vectors. The term effective amount means an amount that can reduce the intensity or stop the development of a disease, degenerative condition or lesion. The effective amount can be determined on an individual basis, taking into account the existing symptoms and expected results. One skilled in the art can determine an effective amount, taking into account the above factors, within the limits of routine experimentation.

According to preferred gene therapy methods, an effective amount of an interferon polynucleotide sequence located in a concomitant vector (i.e., a carrier) can be injected directly into the target cell or tumor tissue as a direct injection using a needle or catheter or other tube that is placed in the tumor or into the blood vessel that feeds this tumor. The magnitude of the dose depends primarily on factors such as the nature of the disease, the chosen interferon, age, body weight and health status of the needy subject, and therefore may be different for different subjects. When using the vector for gene therapy, it is assumed that an effective amount for a person is in the range from about 0.1 to about 10 ml of physiological saline containing from about 1x10 ⁷ to about 1x10 ¹² plaque forming units (PFU) / ml expressing viral vectors including interferon.

As described above, gene ΙΕΝ can be introduced by direct injection into solid tumors. Alternatively, an injection can be made into a blood vessel that feeds this tumor. In addition, parenteral administration of this vector is possible. For the introduction of polynucleotides encoding interferon, you can use local, intraocular, parenteral, nasal, intratracheal, intrabronchial, intramuscular, subcutaneous and other methods of administration. Parenteral administration includes intravenous, intramuscular, intraperitoneal and subcutaneous administration. A more complex approach may be the parenteral administration of a virus or chemical conjugate, which is localized in a tumor of a certain type, due to natural tropism or the presence of a surface molecule that binds to a receptor present only in certain types of tumors.

As described above, the present invention relates to methods for producing a cell expression system for expressing a gene product (e.g., interferon) in a mammalian recipient, the expression system thus obtained and pharmaceutical compositions containing said expression system. The following examples show the feasibility of cell gene therapy in animal model systems.

Example 1. A typical animal model.

Polynucleotides capable of expressing interferon typically experience ίη νίνο in an animal model. In mice, tumor cells cause tumor formation by injecting them with human tumor cell lines. Naked mice (pi / pi line) are immunodeficient and do not reject foreign tumor cells. Tumors form within 1-2 weeks after the injection of tumor cells, although the exact period of time depends on the number of injected cells and oncogenic properties of cell lines. Interferon expressing polynucleotides are introduced into the tumor cells by conventional culture transfection methods prior to their injection into mice. Alternatively, the corresponding polynucleotide can be introduced into the tumor by transfection or viral transduction ίη νίνο after the formation of tumors in mice.

An example is a study in which cells belonging to the NTV9 bladder cancer cell line (Niamd e1 a1., See above) or to the AEB1-B27 retinoblastoma cell line (Takayaky e1 a1, Proc. Ναΐ Asal. 8c1) are used I8A, 88: 5257-5261, 1991). To deliver an isolated polynucleotide encoding interferon that is in culture, tumor cells are transfected (using conventional methods, such as precipitation with calcium phosphate, electroporation or transfection with lipofectamine) or directly infected (using retrovirus, adenovirus, baculovirus or adeno-associated virus). In the case of an effective virus infection, in which 100% of the cells successfully acquire the corresponding polynucleotide, it is not necessary to select cells. In addition, it was established (Example 3) that only a small number of cells in percentage terms should express the interferon gene, therefore, drug selection is usually not required.

When performing selection in the case of transfection, which is not as effective as virus infection, the cells are transfected with an expression vector encoding a drug resistant gene (such as neogen that encodes resistance to 0418) and a polynucleotide, such as the interferon gene. Control transfection is performed only with a vector encoding a drug resistant gene.

After approximately 2-3 weeks of selection in the medium containing the drug, the cell colonies are collected and injected subcutaneously into the flank of female (gsLe) female PC / PC mice at a cell concentration of about 10 ⁶ in a volume of about 100 μl. Virus-infected cells are injected without performing a selection step. Mice are monitored for at least 2 months and the tumor size is measured weekly with a caliper.

Alternatively, non-transfected or uninfected tumor cells are injected subcutaneously into the flank of mice. After the tumor is formed, DNA or a virus containing a polynucleotide encoding interferon is injected directly into the tumor. Many viruses are suitable for this method, although recombinant adenoviruses are the most effective, and the advantage of recombinant retroviruses is that they stably integrate into the genome of the tumor cell. DNA can be introduced into cells as an injection of a mixture of DNA with cationic liposomes. DNA or viruses lacking the interferon gene are injected into the tumors of other mice that serve as control animals. The development or reduction of the tumor is controlled with a caliper.

Example 2. A typical model of a malignant tumor of the lung.

An example is described below of a method for treating a small cell malignant tumor of a human lung with an isolated polynucleotide encoding interferon encapsulated in liposomes, according to which a polynucleotide encoding interferon is introduced into cells requiring such treatment using liposomes, for example, in the form of finely atomized liposomes . The polynucleotide encoding interferon administered as an aerosol is uniformly deposited on the surface of the nasopharynx, tracheobronchial tree and in the pulmonary region. See Kpshy apb Sybey, Eig. b. S1sh. Μίοτο, apb 1pGss (. Όίδ., 7: 721-731 (1988) for familiarization with liposomal aerosols. For the treatment of malignant lung tumors in this way, the polynucleotide encoding interferon is purified by any known method. The polynucleotide encoding interferon is mixed with liposomes and introduced with a high degree of effectiveness, since aerosol delivery is non-irritating and well tolerated by volunteers and patients, the polynucleotide encoding interferon-containing liposomes is easily administered to patients suffering from a malignant tumor about at any stage. The liposomes are administered using nasal inhalation or endotracheal tube connected to a nebulizer at a dose sufficient to inhibit tumor growth. The aerosol is administered daily for two weeks, after which, if necessary, repeat the treatment.

Ίη νίνο studies on the treatment of orthotopic small cell lung cancer can be performed using a tumor that is injected into the right main bronchus in athymic nude (pi / pi) mice (approximately 1.5x10 ⁶ cells / mouse). After 3 days, they begin treatment of mice (daily for 3 consecutive days), which are injected endobronchially with the interferon gene encapsulated in liposomes and the presence of interferon gene sequences is checked. The formation of the tumor and its size are controlled, for which the tumor is measured with a caliper and the survival of the mice is evaluated.

Example 3. Gene therapy ex νίνο betainterferon 1a gene.

In this example, a human breast cancer cell line, ΜΒΆ-ΜΌ-468 (obtained from the American Type Culture Collection) was used. Cells do not infect or infect adenovirus expressing the human beta interferon 1a gene. In this case, the adenovirus removed the E1 genes and there is a temperature-dependent mutation in the E2a gene. Methods of obtaining this adeno virus are described in the work of EpdeShagb! e1 a1., (1994), Sepe Tietaru, 5: 1217-1229 (for a more detailed description, see also below). Briefly, the essence of these methods can be summarized as follows: the beta-interferon 1a gene is preliminarily cloned in the pABSMU1shk1 adenovirus vector so that the SMU 1E1 promoter stimulates gene transcription, resulting in an adenovirus transfer plasmid. A gene is inserted into this vector instead of the deleted E1 gene. Recombinant adenovirus with this interferon gene is obtained by recombination of the transfer plasmid and the adenovirus genome in 293 cells. The virus is subjected to hemolytic purification and titrated according to plaque assays by conventional methods.

Materials and methods

Cell culture. Human malignant cells ΜΌΑ-ΜΒ-468, Niy7, KM12LA4, ME180, HeLa, I87 and 293 are placed as adhesive cultures in the Dulbecco modified Eagle medium containing 10% bovine serum, 2 mM glutamine, penicillin and streptomycin, and essential amino acids vitamins.

The production of purified adenoviruses. The adenovirus transfer vector encoding the human ΙΕΝβ gene, stimulated by the early cytomegalovirus promoter, pbCΜVΙιιιΙΕΝΙΕΝβ, is constructed by ligation of a cDNA insert encoding human β-β 1a into plasmid pABCMU1tk1 (see Epidebat, e1 a1, 1994). Plasmid rABSMU-11i1E ^ is cotransgenically transferred to 293 cells with genomic DNA purified from temperature-dependent adenovirus H51M25. Recombinant adenoviruses isolated from individual plaques are used to infect 293 cells at 39 ° C, after which the supernatants are tested for expression of the β-gene by enzyme-linked immunosorbent assay (EIA8A). Adenoviruses carrying ΙΕΝ-β cDNA (Η5.11Η1ΙΕΝβ) are identified and then amplified. A temperature-dependent adenovirus encoding a β-galactosidase colorimetric marker (H5.1101ac2) is similarly prepared. Viral preparations are produced in 293 cells and purified with CgC1 gradients after two plaque separation cycles. In these preparations, the presence of wild-type adenovirus was not detected.

Subconfluent cells infect H5.110LPE ^ in accordance with a multiplicity of infection (ΜΟΙ) of 100 in 3 ml of medium containing 2% bovine serum. After 15-18 hours, supernatants are collected and quantitatively determined for концентрацииβ concentration by ΕΜδΑ analysis.

Analysis of E8A. 96-well plates are coated overnight at 4 ° C with anti-antibody

ΙΕΝβ person, V02 (§ishshy RyattaseysaI

Japan). The antibody is used in an amount of 10 μg / ml in a surface sensitization buffer containing 50 mM sodium bicarbonate / carbonate, 0.2 mM MDCl ₂ and 0.2 mM CaCl ₂ (pH 9.6). The plates are then blocked with 1% casein in physiological saline with phosphate buffer (PB8) for 1 h at room temperature and samples of ΙΕΝ-β protein standards (Ανο ^ χ ™, B ^ οdeη) diluted with 1% casein and 0.05% are added tween-20. The plates are then sequentially incubated at room temperature for 1 hour with anti-β-rabbit serum (dilution 1: 2000), for 1 hour with anti-rabbit donkey antibody conjugated to horseradish peroxidase (NKR) (Zatti Itti Kekeagsy, division 1 : 5000), and a substrate solution (4.2 mM TMB and 0.1 M sodium acetate and citric acid, pH 4.9). The reaction is stopped 2n. a solution of H ₂ 8O ₄ and measure the optical density at 450 nm.

Experiments using mice. Female pi / pi Ba1b / c mice aged 4-6 weeks were provided by Tatoshs Gagp (Boston, Massachusetts). All mice were kept in a sterile animal box Vüdeg1 for at least 2 weeks before the start of each experiment. For experiments ex νίνο, infected and uninfected cells are harvested using trypsin / ΕΌΤΑ solution and washed twice with PB8. These cells are mixed immediately prior to injection into mice in the following respects. A total of 2x10 ⁶ cells in 100 μl of PB8 are implanted subcutaneously in mice in the right flank. Using a caliper, measure the length and width of the tumor and express it as the average diameter of the tumor (mm).

For experiments on direct injection of ίη νίνο (Example 4), 2 × 10 ⁶ tumor cells in 100 μl of PB8 are implanted subcutaneously in the mice of HbBe. When the tumor size reaches 5-6 mm in diameter, 100 μl of PB8 containing different doses of recombinant adenoviruses is injected into the center of the tumor as a single injection. Using a caliper, measure the length and width of the tumor. Tumor size is calculated by averaging the length and width. The death of animals was ascertained at the time of killing of mice that showed signs of bleeding or the tumor size reached 10% of the total body weight. Apoptosis is examined using the Apoptosis Detection Kit ίη 8Yi provided by Opsog, Schc. (catalog number 87110-ΚΙΤ).

results

First, transduction efficiency and transgene expression by adenoviral vectors were determined. MOA-MB-468 human breast cancer cells infected H5.1101ac2 with an increasing multiplicity of infection (ΜΟΙ). After staining with X-da1, it was found that at ΜΟΙ equal to 100, the efficiency of gene transduction in these cells reaches approximately 100% (data not shown). Thus, cells of a malignant tumor of the breast are infected in culture in relation to 100 plaque forming units (PFU) per cell, as our experience with these malignant cells shows that this is the smallest virus: cell ratio that leads to gene expression in each cell populations.

During the first experiment, 18 hours after infection, mice of HBs were injected subcutaneously in the back with 2x10 ⁶ cells. Five mice were injected with uninfected cells and five mice were injected with cells infected with adenovirus-β. All mice that were injected with control uninfected cells formed tumors of significant size. Tumors were absent in all mice injected with cells treated with adenovirus expressing β-β (H5.110NE ^: Table 1).

To exclude the possibility that exposure to воздействиеη νίίτο tumor cells with ΙΕΝ-β protein may lead to a loss of ίη νίνο tumorigenicity, MOA-MB-468 cells were treated with ΙΕΝ-β protein at a concentration detected 18 hours after virus infection. After thoroughly washing the cells, 1'SBE mice were injected with an equal number of treated cells, untreated cells, or a mixture containing 10% of the treated cells. Tumors developed in all three groups of mice (data not shown), suggesting that delivery of the ΙΕΝ-β ex νίνο gene, rather than protein treatment with ίη νίΐτο, is important for suppressing tumor formation.

To determine whether malignant cells expressing the β-gene can cause destruction of non-transduced cells, the following experiment was performed. The same malignant cells were not infected, infected with an adenovirus expressing the β-gene (H5.110ΗΙΕΝβ), or infected with the same type of adenovirus, but expressing the 1ac2 gene (H5.1101ac2), which encodes a β-galactosidase reporter protein that does not have an anti-cancer effect and therefore used to control any effects caused by the adenovirus itself. Cells were infected with adenovirus in relation to PFU: a cell of 100. Tumor cells MOA-MB-468 were separately infected with SHOWER ¹ ^ or H5.1101ac2 with MOF of 100. 18 hours after infection, infected cells were harvested and some of these cells were mixed with uninfected cells before injection into mice. The same number of infected cells, uninfected cells, or a mixture containing 10% of infected cells and 90% of cells not treated with the virus were subcutaneously implanted with pi6c Ba1b / c mice. Tumor growth was monitored after twenty days. All mice that were implanted with uninfected cells had tumors, and mice that were injected with 100% of cells infected with Η5.110ΜΡΝβ or H5.1101ac2 had no tumors, indicating that infection with any virus could suppress oncogenicity (Table 1 ) However, all mice that were injected with 10% of cells infected with H5.1101ac2 had tumors, while all mice that were injected with 10% of cells infected with H5.1101ac2 had no tumors. Based on this, it can be concluded that although H5.1101ac2 infection inhibits the formation of a tumor of infected cells, this drug is not able to block the oncogenicity of co-injected naive or uninfected cells. In contrast, transduction of H5.110YPH in 10% of the cells was sufficient to suppress the oncogenicity of cells transduced by the virus, as well as those cells that were not transduced. Tumor inhibition in 100% of populations Table 1

Sample Average diameter] p of the tumor (with mm) 12th day The 19th day 26th day 33rd day 100% uninfected cells 4.1 ± 0.6 4.9 ± 0.8 5.9 ± 0.5 6.3 ± 0.6 100% A6U-1RX 0,0 0,0 0,0 0,0 10% A6U-1RX 0,0 0,0 0,0 0,0 3% AbU-! P \ 0,0 0,0 0,0 0,0 1% AbU-! P \ 0,0 0,0 0,0 0,0 0.3% AbU-! P \ 2.8 ± 0.2 2.9 ± 0.3 3.0 ± 0.6 1.8 ± 2.0 100% AbU-1as2 0,0 0,0 0,0 0,0 10% AbU-1as2 4.8 ± 0.4 5.2 ± 0.6 6.2 ± 0.5 6.5 ± 0.5 3% AbU-1as2 4.3 ± 0.5 4.6 ± 0.6 5.8 ± 0.8 6.5 ± 1.0 1% AbU-1as2 4.3 ± 0.4 4.6 ± 0.4 5.0 ± 0.5 6.3 ± 0.9 0.3% AbU-1as2 4.4 ± 0.5 4.7 ± 0.6 No data No data

Tumor development was completely blocked in mice that were injected with only 1% of cells transduced with H5.110PHB (Table 1). During the first week after injection of the cells, very small tumors (they were palpable, but not large enough to be measured) were found in mice that were injected with 10, 3 and 1% of cells with H5.110111 ΡΝ β. However, all these small tumors completely disappeared by the 9th day. Based on this, it can be assumed that some cells remained viable for a short period of time and expressed the β-gene over this period, which caused the death of the entire tumor. In a transduction of H5.1101ac2, it can occur due to some general toxic effects or some antitumor effects of this generation of adenovirus, but it should be noted that transduced cells have the ability to replicate ίη igo (unpublished data).

To determine the amount of interferon-containing virus necessary for suppressing oncogenicity, tumor cells infected with H5.110YPHB and H5.1101ac2 were separately mixed with uninfected cells in different ratios, provided that in all cases there was an excess of uninfected cells. Mixing was performed in such a way that different samples contained only 10, 3, 1, 0.3% of cells infected with adenovirus, while the remaining cells were not infected. Immediately after mixing, these cells were injected with pi6c mice. The results are shown in table. 1. The diameters of the tumors were measured in two directions at different times after injection of the cells. All values are given in table. 1, represent the mean ± standard deviation of diameter measurements in the transverse and longitudinal directions in 4 mice. Measurements were performed on 12, 19, 26 and 33 days after injection of tumor cells.

In several experiments performed with the introduction of this cell line to pi6c mice, the tumor did not appear at all when 1% of the cells were transduced with H5.1101ΙΡΝβ, and there was 100% animal survival (data not shown). Tumors were formed in mice injected with 0.3% of cells transduced with H5.110YPHB, however, the size of these tumors was significantly smaller than in control mice, and 2 of 5 mice in the 0.3% group had tumors completely disappeared on the 33rd day (table. 1).

In contrast, in mice that were injected with from 10 to 0.3% of the cells treated

H5.110.1ac2, tumors of the same size were formed as in the uninfected group, and in none of these control groups the reverse development of the tumor was observed (Table 1).

Obviously, the expression of human beta-interferon (ΜΕΝ-β), even in a very small percentage of cells, blocks oncogenicity in gbe mice. Then, a study was carried out to determine the smallest ratio of cells infected with H5.110NE ^ necessary to affect the tumor, which is not necessarily expressed in its blocking, and increase the survival of mice. The same number of имп-ΜΒ-468 cells containing 0.3, 0.1, 0.33, 0.01 and 0% of cells infected with Η5.1101ιΙΕΝβ were implanted in Pibe mice, and tumor growth was monitored. Mice that were injected with 0.3 or 0.1% of infected cells had much smaller tumors than mice that had only uninfected cells (Fig. 1A). Of the tumors that formed at 0.3 and 0.1% transduction, respectively, 3 out of 5 tumors and 1 out of 5 tumors completely disappeared. A significantly longer survival of mice was observed in groups with 0.3 and 0.1% transduction. All animals that were implanted with 0, 0.01 or 0.03% of infected cells died within 75 days, while in 0.1 and 0.3% groups, respectively, 1 out of 5 and 3 out of 5 animals were alive and had no tumors on the 109th day when this experiment was completed (Fig. 1B).

It is likely that only a small fraction (more than 0.3%, preferably more than 1.0%) of all tumor cells needs to be transfected or infected in order to obtain an effective result. This conclusion differs from such approaches to antitumor gene therapy as delivery of wild-type tumor suppressors (for example, p53), in which all cells in the tumor must have the tumor suppressor gene.

Thus, the interferon gene shows a strong antiproliferative effect of ίη νΐνο after infection with ίη νίίτο. Control experiments show that this is due to interferon, not adenovirus. Gene therapy with β-interferon ex νΐνο of other xenograft human tumors

The transduction effect of Η5.1101ιΙΕΝβ was investigated in relation to other types of tumor cells in the ex νΐνο human xenograft model. Human colon cancer cells ΚΜ12Ό4оли, human liver cancer cells Ηιι1ι7, or human cervical cancer cells ME180 transduced Η5.1101ιΙΕΝβ. The same number of cells containing 10, 1, or 0% transduced cells was tested with respect to their ability to form tumors in gbe mice. Injection of uninfected cells of all three types caused the formation of rapidly growing tumors in all mice. In contrast, the administration of ex νΐνο 10% of the cells infected with Η5.1101ιΙΕΝβ caused either a complete suppression of tumor development or a later appearance of slowly growing tumors in all the animals studied (Fig. 2 A). In contrast to the results obtained using ΜΌΑ-ΜΒ-468 cells, in which 1% transduction of Η5.1101ιΙΕΝβ completely prevented tumor formation, 1% transduction of these three types of cells caused the formation of tumors, although they were smaller than in uninfected control animals in each period of tumor measurement. Mice that were injected with 10 and 1% of transduced cells had a longer survival time compared with mice that were injected with uninfected cells (Fig. 2B). Thus, adenovirus-mediated delivery of ex νΐνο of the ΙΕΝ-β gene to many different tumor cells causes an effective suppression of oncogenesis and leads to an increase in the survival time of animals.

Example 4. Gene therapy ίη νίίτο betainterferon 1a gene.

Instead of ex νΐνο, gene therapy ίη νΐνο can be performed. In the case of ίη νΐνο gene therapy, the gene is administered directly to the patient. In this example, an adenovirus with the human β-β gene (H5.110OE ^) was injected directly into a solid tumor. That is, the cells of a malignant tumor of the human mammary gland ΜΒΑ-ΜΌ-468 in an amount of 2 × 10 ^{6 were} injected subcutaneously in the back of 50 mice of the fur. Subcutaneous tumors with a diameter of about 5-6 mm were formed in gscheb mice 24 days after subcutaneous injection of ΜΌΑ-ΜΒ-468 cells. During this time period, the tumors were treated with ΡΒ8 or вектора5.110ΕΙΕΝβ and Η5.1101; · ιοΖ vectors at different doses of the virus from 1x10 ⁷ to 3x10 ⁹ PFU / mouse as a single intratumoral injection.

The data shown in FIG. 3 show that within 14 days the introduction of a single dose of Η5.110ΕΙΕΝβ at a concentration of 3x10 ⁹ , 1x10 ⁹ or 3x10 ⁸ total PFU causes a decrease in the tumor. Complete regression of tumor occurs in 4 of 5 mice per group, received 3x10 ⁹ pfu, and in 3 of 5 animals per group, received 1x10 ⁹ pfu. In tumors in which 1x10 ⁹ PFU Η5.1101ιΙΕΝβ was injected, a high local concentration of ΙΕΝ-β of approximately 1500 IU / ml can be detected, while in the serum, the concentration of ΙΕΝ-β is only 37 IU / ml. Lower doses of Η5.110ΕΙΕΝβ, equal to 1x10 ⁸ , 3x10 ⁷ and 1 x ^{10 7} PFU, have little or no effect at all (Fig. 3 and data are not shown), indicating that the antitumor reaction depends on the dose. Injections of PB8 or H5.1101ac2 in equivalent doses do not cause tumor regrowth (Fig. 3). When observing tumors for a longer period of time, growth retardation and the reverse development of some individual tumors, in which H5.1101ac2 was injected at a concentration of 3x10 ⁹ PFU, can be detected, which suggests that the control virus at this dose can slightly inhibit tumor growth . Treatment with H5.1101ιΙΕΝβ with a concentration of 3x10 ⁹ , 1x10 ⁹ or 3x10 ⁸ PFU significantly increases the survival of mice compared to mice that were injected with PB8 or H5.1101ac2 (data not shown). In addition, a treatment regimen was studied consisting of several injections, in particular, 5 injections of H5.110LBYv at a dose of 1x10 ⁸ PFU, which were done every three days in the MOL-MV-468 and HeLa tumors, as a result of which growth was slowed down and reverse development of tumors of both types (unpublished data). A similar experiment, ίη νινο, was performed using the I87 human glioma cell line. These cells were very sensitive to β-β, since the complete reverse development of the tumor was observed in 4 of 4 mice that were injected with H5.110LBYv in a dose of 1 × 10 ⁹ PFU, and in 2 of 4 mice that were injected with 1 × 10 ⁸ PFU (data not shown ) These results suggest that direct and local delivery of ίη νινο adenovirus containing the ΙΕΝ-β gene can cause significant antitumor effects.

4 days after injection of 1x10 ⁹ PFU of the tumor virus, MOL-MB-468 was taken for histological examination. During this period of time, the tumors into which H5.110B3B was injected showed signs of reverse development. Histological analysis of MOL-MB-468 tumors was performed by hematoxylineosin staining. A greater number of apoptotic cells were found in the tumor into which H5.110LBw was injected than in the tumor into which H5.1101ac2 was injected. The presence of apoptosis was confirmed by direct fluorescence detection of fragmented genomic DNA labeled at the ends. Very few infiltrating mononuclear cells were found in tumors that were injected with H5.110LBH or H5.1101ac2, which indicates that the cellular immune response is not important for the reverse development of the tumor under the action of H5.110Blb in this model.

In the experiments described above, ex νινο and ίη νινο, only the direct antiproliferative effect of interferon was studied. Since mice with immunodeficiency were used in the studies, any immunostimulating activity that interferon possesses and which can stimulate tumor destruction should be absent. In addition, since interferons are not intended for interspecific crossbreeding between humans and mice, human ΙΕΝ-β used to suppress human malignant tumor cells in these mice apparently cannot inhibit angiogenesis since human ΙΕΝ-β does not act on endothelial cells vessels. Therefore, it is entirely possible that when adenovirus is administered to a person with human β-β, an even more pronounced antitumor effect will take place. This action can be modeled on immunocompetent primates other than humans. Alternatively, adenovirus with the ΙΕΝ-β gene of mice can be introduced into immunocompetent mice with tumors of mouse origin. There are many tumor models of syngeneic mice.

The data presented here demonstrate the remarkable ability of β-interferon gene therapy to block the formation of tumors without ηονο, as well as cause the reverse development of existing tumors. Ex νινο transduction experiments confirm that the administration of a potent secreted protein in only 0.3-1.0% of transduced cells blocks the formation of MEL-MB-468 tumors. A number of other tumor cell lines were tested and, although there were differences in the action of ΙΕΝ-β, all tumors could suppress 1-10% of the cells transduced with ΙΕΝ-β. Given that in order to suppress tumor growth, a relatively small percentage of cells secreting β-β is necessary, direct intratumoral injection of adenovirus into previously formed tumors was performed. In this case, the ΙΕΝ-β gene also had a strong effect, and partial or complete reverse development of tumors was observed as a result of individual injections of the virus.

In these studies, the significant reverse development of tumors, apparently, was primarily the result of the direct antiproliferative or cytotoxic effect of ΙΕΝ-β. This conclusion is supported by the fact that the ΙΕΝ-β gene used in this study is of human origin, and human ΙΕΝ-β is not involved in a cross-reaction with mouse host cells. In addition, the used mice with immunodeficient gbe cells lack T-lymphocytes, the main effector cell in interferon-induced immunostimulation type 1 (ΤοιΤβΐι, Ό.Ε. еί а1., (1996), 8c1eg1ce, 272: 1947-1950 and Kidde, b. ί a1., (1997) I. Exp.Meb., 185: 825-831). In addition, in rapidly developing cells after the introduction of the β-β gene, there is no obvious increase in the infiltration of mononuclear cells. These results confirm the view that the antiproliferative effect mediated by β-interferon may itself be sufficient to stimulate the reverse development of the tumor. Our data are compatible with the clinical correlation that was previously detected between the sensitivity of ίη νίίτο malignant cells to ΙΕΝ-induced antiproliferative activity and the therapeutic effect of ίη νίνο (ΕίηΗοτη aib Cragel (1996) ί. Ιη ^ ΓίοϊΌη Sufkshe 27 Keke 2: 28. )

In short, it has been found that adenovirus-mediated β-interferon gene therapy can have an effective antitumor effect on mice used as an animal model. The introduction of ex νίνο of the ΙΕΝ-β gene in a very small percentage of cells is sufficient to suppress the formation of a tumor, and direct intratumoral injection of a single dose of the ΙΕΝ-β gene leads to the reverse development of tumors. Without binding itself to any theory, we can conclude that a strong antitumor effect can be the result of the autocrine and paracrine action of β-interferon. This antitumor effect may be important for further gene therapy studies of malignant tumors, in which the degree of gene delivery is apparently a limiting factor and it is necessary to achieve a large imitation effect. Therefore, local β-interferon gene therapy is a promising treatment strategy for tumors in humans.

Modifications

Obviously, the foregoing is only a detailed description of specific preferred embodiments of the invention. Therefore, specialists in this field should be clear that the scope of this invention includes various modifications and variations.

Claims (22)

1. A method of treating malignant tumors using ίη νίνο gene therapy, comprising the step of administering to the subject a therapeutically effective amount of a viral vector, characterized in that said vector contains a gene encoding β-interferon, and wherein said vector is selected from the group consisting of an adenoviral vector, lentiviral a vector, a vector derived from the Epstein-Barr virus, a baculovirus vector, and a vector derived from the herpes simplex virus. 2. The method according to claim 1, characterized in that the vector is selected from the group consisting of an adenoviral vector, a baculovirus vector and a lentiviral vector. 3. The method according to claim 1, characterized in that the vector is an adenoviral vector. 4. The method according to claim 3, characterized in that the adenoviral vector contains a deletion in the E3 gene. 5. The method according to claim 3 or 4, where the adenoviral vector contains a deletion in the E1 gene. 6. The method according to claim 1, characterized in that the vector is administered by a route selected from the group consisting of direct injection into the tumor, local administration, intraocular administration, parenteral administration, intranasal administration, intratracheal administration, intrabronchial administration and subcutaneous administration. 7. The method according to claim 6, characterized in that the pathway is parenteral administration selected from the group consisting of intravenous administration, intramuscular administration and intraperitoneal administration. 8. The method according to claim 6, characterized in that the vector is administered by direct injection into the tumor. 9. The method according to claim 1, characterized in that the tumor is selected from the group consisting of malignant glioma, melanoma, hemangioma, leukemia, lymphoma, myeloma, colon and rectal cancer, non-small cell carcinoma, breast cancer and ovarian cancer. 10. The method according to claim 9, characterized in that the tumor is a malignant glioma. 11. A method of treating malignant tumors using ίη νίνο gene therapy, comprising the step of administering to the subject a therapeutically effective amount of a replication-defective vector, wherein said vector contains a gene encoding β interferon, and wherein said vector is selected from the group consisting of an adenoviral vector, lentiviral a vector, a baculovirus vector, a vector derived from the Epstein-Barr virus, and a vector derived from the herpes simplex virus. 12. The method according to claim 11, characterized in that the vector is selected from the group consisting of an adenoviral vector, a baculovirus vector and a lentiviral vector. 13. The method according to p. 12, characterized in that the vector is an adenoviral vector. 14. The method according to item 13, wherein the vector is deleted in one or more genomic regions selected from the group consisting of regions Ε1, E2A and E4 of the adenoviral genome. 15. The method according to item 13 or 14, characterized in that the vector contains a deletion in the E3 gene. 16. The method according to 14, characterized in that the vector does not contain the E1 gene. 17. The method according to clause 16, wherein the vector further comprises a deletion in the E3 gene. 18. The method according to claim 11, characterized in that the vector is administered by a route selected from the group consisting of direct injection into the tumor, local administration, intraocular administration, parenteral administration, intranasal administration, intratracheal administration, intrabronchial administration and subcutaneous administration. 19. The method according to p. 18, characterized in that the pathway is parenteral administration selected from the group consisting of intravenous administration, intramuscular administration and intraperitoneal administration. 20. The method according to p, characterized in that the vector is administered by direct injection into the tumor. 21. The method according to claim 11, characterized in that the tumor is selected from the group consisting of malignant glioma, melanoma, hemangioma, leukemia, lymphoma, myeloma, colon and rectal cancer, non-small cell carcinoma, breast cancer and ovarian cancer. 22. The method according to item 21, wherein the specified tumor is a malignant glioma.